An isolated interleukin-34 polypeptide for use in preventing transplant rejection and treating autoimmune diseases

ABSTRACT

The invention relates to an isolated interleukin-34 (IL-34) polypeptide for use in preventing or treating graft rejection, autoimmune disease, unwanted immune response against therapeutic proteins and allergy. The invention also provides an in vitro method for determining whether a patient is at risk of transplant rejection, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies, comprising a step of determining the expression level of IL-34 in a biological sample obtained from said patient, wherein the presence of IL-34 is indicative of a reduced risk of transplant rejection, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies.

FIELD OF THE INVENTION

The invention is in the field of immunotherapy. More particularly, theinvention relates to an isolated interleukin-34 (IL-34) polypeptide foruse in preventing or treating transplant rejection, autoimmune disease,unwanted immune responses against proteins expressed in the course ofgene therapy and/or therapeutic proteins (such as factor VIII,antibodies, etc) and allergy.

BACKGROUND OF THE INVENTION

Induction of immune tolerance is a powerful tool to control immuneresponses responsible for pathological situations. Cytokines, enzymescontrolling metabolic pathways and cell surface molecules capable ofinducing tolerance have been described. Despite these findings, evidencefor other non-identified mechanisms exists and is thus important toidentify new mediators of immune tolerance.

Organ transplantation has shown very significant improvements in bothprevention and treatment of acute rejection but subclinical episodes andchronic graft dysfunction still heavily impact medium and long-termgraft survival (1). Emerging therapeutic strategies, among themtolerance induction to donor antigens are moving into the clinics afteryears of experimental models work (2, 3). Among natural mechanisms andtolerance inductive strategies, different types of regulatory cells areamong the most promising ones (4). CD8⁺ regulatory T cells (CD8⁺ Tregs)in the transplantation field but also in other pathophysiologicalsituations have been highlighted in recent years (5-8). Cytokines,enzymes controlling metabolic pathways, and cell surface molecules,capable of inducing tolerance, have also been described as new mediatorsof immune tolerance.

A new cytokine called IL-34 was identified in 2008 (9). Studies showedthat IL-34 shares homology with M-CSF and acts through a commonreceptor, CD115 also called CSF-1R (9) expressed on the cell surface ofmonocytes, and in the brain through a new described receptor,Receptor-type Protein-tyrosine Phosphatase ζ (PTP-ζ) (10). However,studies have demonstrated that IL-34 and M-CSF displayed distinctbiological activity and signal activation (11), probably due todifferential spatial and temporal IL-34 and M-CSF expression (12). IL-34function has been mainly related until now with monocytes andmacrophages (osteoclasts, microglia), as well as DCs, survival andfunction (12). Datas on the expression of IL-34 in resting cells werepartially overlapping since mice with GFP under the control of the IL-34promoter showed positive keratinocytes, hair follicles, neurons,proximal renal tubule cells and seminiferous tubule germ cells (12),whereas mRNA and protein analysis showed heart, brain, lung, liver,kidney, spleen, thymus, testicles, ovaries, prostate, colon, and smallintestine, and abundant protein expression in spleen red pulp andosteoclast (9). Upon inflammation, other cells such as fibroblasts (13),osteoclasts (14) and articular synovial cells (13) upregulated IL-34expression. So far, expression of IL-34 by other lymphoid cells andparticularly T cells has not been described or demonstrated. Similarly,IL-34 has not been linked to effects on immune function of DCs or Tcells since the decreased response to DTH antigens or CNS viralinfections in IL-34-deficient mice was linked to paucity of Langerhan'sand microglia, in skin and CNS respectively (12). Finally, there is nodescription to date of a role for IL-34 in tolerance in transplantation.

In a model of cardiac allograft in rats, it has been previously shownthat blocking of CD40-CD40L, by CD40Ig treatment, induces long termgraft survival through generation of of CD8⁺CD45RC^(low) Tregs (termedCD8⁺CD40Ig Tregs) (15). It also been showed that these CD8⁺ Tregs imposeallogeneic tolerance partially through production of IFNγ andfibrinogen-like protein 2 (FGL2) (15, 16), and recognition of a dominantMHC-II-derived donor peptide (17). A potential role for FGL2 as animmune tolerogenic mechanism was first suspected when pan-genomictranscriptomic comparison of CD8⁺CD40Ig Tregs vs. CD8⁺CD45RC^(low) Tregsfrom naïve rats showed increased FGL2 expression (16). The sametranscriptomic analysis revealed that IL-34 was overexpressed in inCD8⁺CD40Ig Tregs from long-term recipients vs. CD8⁺CD45RC^(low) Tregsfrom naive animals.

Therefore, despite considerable advances in prevention of transplantrejection, such pathology remains associated with high morbidity andmortality and there is a desperate need for new mediators of immunetolerance and new tolerance inductive strategies (more particularly bydown-regulating T-cell responses) for use in the prevention or thetreatment of transplant rejection (or for use in the induction oftransplant tolerance) as well as autoimmune disease, unwanted immuneresponses against proteins expressed in the course of gene therapyand/or therapeutic proteins and allergy.

Moreover, there is a need for an easily measurable biomarker predictingthe risk of transplant rejection. Such a biomarker would thus be usefulfor monitoring transplanted patients and also adjusting theirimmunosuppressive treatment.

Until now, no study has examined whether IL-34 might induce immunetolerance and predict whether a transplanted patient is tolerant or not(displaying thus an increased risk of transplant rejection and thereforerequiring an appropriate immunosuppressive treatment). Similarly, IL-34has not been linked to effects on immune function of DCs or T cells andits suppressive potential in transplantation has never been suspectedand studied.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an isolated Interleukin-34(IL-34) polypeptide or a polynucleotide encoding therefor for use ininducing immune tolerance in a patient in need thereof.

In a second aspect, the invention relates to an isolated IL-34polypeptide or a polynucleotide encoding therefor for use in preventingor reducing transplant rejection in a patient in need thereof.

In a third aspect, the invention relates to an isolated IL-34polypeptide or a polynucleotide encoding therefor for use in preventingor treating autoimmune diseases, unwanted immune response againsttherapeutic proteins and allergies in a patient in need thereof.

In a fourth aspect, the invention relates to pharmaceutical compositionor a kit-of-part composition comprising an isolated IL-34 polypeptide ora polynucleotide encoding therefor and an immunosuppressive drug.

In a fifth aspect, the invention relates to an in vitro method fordetermining whether a patient is at risk of transplant rejection,autoimmune diseases, unwanted immune response against therapeuticproteins or allergies, comprising a step of determining the expressionlevel of IL-34 in a biological sample obtained from said patient,wherein the presence of IL-34 is indicative of a reduced risk oftransplant rejection, autoimmune diseases, unwanted immune responseagainst therapeutic proteins or allergies.

In another aspect, the invention relates to method for adjusting theimmunosuppressive treatment administered to a patient in need thereof,comprising the following steps of (i) performing the method fordetermining the risk as above defined, and (ii) adjusting theimmunosuppressive treatment.

DETAILED DESCRIPTION OF THE INVENTION

The inventors demonstrated that IL-34 induces immunosuppression,inhibits primary T cell responses and induces T cell tolerance. Thisapproach is of interest in the fields of autoimmunity, allergy,transplantation, treatment with therapeutic proteins and gene therapy,to avoid degradation of self or therapeutic molecules/tissues by theimmune system.

The inventors demonstrated for the first time IL-34 involvement in CD8⁺Tregs immunosuppressive function in vitro. Blockade of its receptorCD115, expressed exclusively on myeloid cells, reverted IL-34suppressive effect on effector CD4⁺ T cells proliferation to pDCs,suggesting an indirect suppression of T cells response via pDCs. Theyshowed for the first time that overexpression of IL-34 inducesregulatory cells in vivo, capable of infectious tolerance. Accordingly,they identified IL-34 as a new mediator of the suppression by CD8⁺Tregs, and as a tolerogenic cytokine efficiently inhibiting allograftrejection.

The inventors also demonstrated that a combination comprising IL-34 anda suboptimal short-term immunosuppressive treatment (rapamycin) enablesa synergistic increase of long-term graft survival vs. each treatmentseparately. They thus evaluated the immunoregulatory potential of thiscytokine in transplantation using AAV-mediated overexpression anddemonstrated a significant prolongation of allograft survival inassociation with a short 10-days course suboptimal dose of rapamycin,leading to 75% of indefinite survival, with total inhibition ofalloantibody production.

The inventors further demonstrated that IL-34 is useful as biomarker forpredicting the risk of transplant rejection.

Therapeutic Methods and Uses

The invention provides methods and compositions (such as pharmaceuticaland kit-of part compositions) for use in inducing and/or maintainingimmune tolerance in a patient in need thereof.

The invention also provides methods and compositions for use inpreventing or reducing transplant rejection in a patient in needthereof.

The invention further provides methods and compositions for use inpreventing or treating autoimmune diseases, alloimmune responses andallergies in a patient thereof.

In a first aspect, the invention relates to an isolated Interleukin-34(IL-34) polypeptide for use in inducing and/or maintaining immunetolerance in a patient in need thereof.

The invention also relates to an isolated macrophage colony stimulatingfactor (M-CSF) polypeptide for use in inducing and/or maintaining immunetolerance in a patient in need thereof.

The invention also relates to a combination of an isolated IL-34polypeptide and of an isolated M-CSF polypeptide for use in inducingand/or maintaining immune tolerance in a patient in need thereof.

As used herein, the term “immune tolerance” refers to a state ofunresponsiveness of the immune system to substances or tissues that havethe capacity to elicit an immune response.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity, in addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages. Immune cells involved in the immuneresponse include lymphocytes, such as B cells and T cells (CD4⁺, CD8⁺,Th1 and Th2 cells); antigen presenting cells (e.g. professional antigenpresenting cells such as dendritic cells); natural killer cells; myeloidcells, such as macrophages, eosinophils, mast cells, basophils, andgranulocytes.

For instance, immune responses are involved in transplant rejection, aswell as in the concomitant physiological result of such immuneresponses, such as for example, interstitial fibrosis, chronic graftarteriosclerosis, or vasculitis. Immune responses are also involved inautoimmune diseases and the concomitant physiological result of suchimmune responses, including T cell-dependent infiltration and directtissue injury, T cell-dependent recruitment and activation ofmacrophages and other effector cells, and T cell-dependent B cellresponses leading to autoantibody production.

Thus, treated patients with an IL-34 polypeptide in comparison withuntreated patients display the following physiological features: a) adecreased level of an immune response (specific or not) (thought to bemediated at least in part by antigen-specific effector CD4⁺ T and CD8+lymphocytes); b) a delay in the onset or progression of a immuneresponse (specific or not); or c) a reduced risk of the onset orprogression of an immune response (specific or not). As used herein, theterm “specific” immune tolerance occurs when immune tolerance ispreferentially invoked against certain antigens in comparison withothers.

By “patient in need thereof” is meant an individual suffering from orsusceptible of suffering from transplant rejection, an autoimmunediseases, alloimmune responses or allergies to be treated. Theindividuals to be treated in the frame of the invention are mammals,preferably human beings.

In one particular embodiment, the patient in need thereof is a patientundergoing transplantation.

In a second aspect, the invention relates to an isolated IL-34polypeptide for use in preventing or reducing transplant rejection in apatient in need thereof.

The invention also relates to an isolated M-CSF polypeptide for use inpreventing or reducing transplant rejection in a patient in needthereof.

The invention also relates to a combination of an isolated IL-34polypeptide and of an isolated M-CSF polypeptide for use in preventingor reducing transplant rejection in a patient in need thereof.

As used herein, the term “preventing or reducing transplant rejection”is meant to encompass prevention or inhibition of immune transplantrejection, as well as delaying the onset or the progression of immunetransplant rejection. The term is also meant to encompass prolongingsurvival of a transplant in a patient, or reversing failure of atransplant in a patient.

Further, the term is meant to encompass ameliorating a symptom of animmune transplant rejection, including, for example, ameliorating animmunological complication associated with immune rejection, such as forexample, interstitial fibrosis, chronic graft atherosclerosis, orvasculitis.

As used herein, the term “transplant rejection” encompasses both acuteand chronic transplant rejection. “Acute rejection” is the rejection bythe immune system of a tissue transplant recipient when the transplantedtissue is immunologically foreign. Acute rejection is characterized byinfiltration of the transplant tissue by immune cells of the recipient,which carry out their effector function and destroy the transplanttissue. The onset of acute rejection is rapid and generally occurs inhumans within a few weeks after transplant surgery. Generally, acuterejection can be inhibited or suppressed with immunosuppressive drugssuch as rapamycin, cyclosporin, anti-CD40L monoclonal antibody and thelike. “Chronic transplant rejection” generally occurs in humans withinseveral months to years after engraftment, even in the presence ofsuccessful immunosuppression of acute rejection. Fibrosis is a commonfactor in chronic rejection of all types of organ transplants.

The term “transplantation” and variations thereof refers to theinsertion of a transplant (also called graft) into a recipient, whetherthe transplantation is syngeneic (where the donor and recipient aregenetically identical), allogeneic (where the donor and recipient are ofdifferent genetic origins but of the same species), or xenogeneic (wherethe donor and recipient are from different species). Thus, in a typicalscenario, the host is human and the graft is an isograft, derived from ahuman of the same or different genetic origins. In another scenario, thegraft is derived from a species different from that into which it istransplanted, including animals from phylogenically widely separatedspecies, for example, a baboon heart being transplanted into a humanhost.

In one embodiment the donor of the transplant is a human. The donor ofthe transplant can be a living donor or a deceased donor, namely acadaveric donor.

In one embodiment, the transplant is an organ, a tissue, or cells.

As used herein, the term “organ” refers to a solid vascularized organthat performs a specific function or group of functions within anorganism. The term organ includes, but is not limited to, heart, lung,kidney, liver, pancreas, skin, uterus, bone, cartilage, small or largebowel, bladder, brain, breast, blood vessels, esophagus, fallopian tube,gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limbincluding upper and lower, spleen, stomach, testes, thymus, thyroid,trachea, ureter, urethra, uterus. As used herein, the term “tissue”refers to any type of tissue in human or animals, and includes, but isnot limited to, vascular tissue, skin tissue, hepatic tissue, pancreatictissue, neural tissue, urogenital tissue, gastrointestinal tissue,skeletal tissue including bone and cartilage, adipose tissue, connectivetissue including tendons and ligaments, amniotic tissue, chorionictissue, dura, pericardia, muscle tissue, glandular tissue, facialtissue, ophthalmic tissue.

In a particular embodiment, the transplant rejection is cardiacallotransplant rejection.

As used herein, the term “cells” refers to a composition enriched forcells of interest, preferably a composition comprising at least 30%,preferably at least 50%, even more preferably at least 65% of saidcells.

In certain embodiments the cells are selected from the group consistingof multipotent hematopoietic stem cells derived from bone marrow,peripheral blood, or umbilical cord blood; or pluripotent (i.e.embryonic stem cells (ES) or induced pluripotent stem cells (iPS)) ormultipotent stem cell-derived differentiated cells of different celllineages such as cardiomyocytes, beta-pancreatic cells, hepatocytes,neurons, etc. . . .

In one embodiment, the cell composition is used for allogeneichematopoietic stem cell transplantation (HSCT) and thus comprisesmultipotent hematopoietic stem cells, usually derived from bone marrow,peripheral blood, or umbilical cord blood.

HSCT can be curative for patients with leukemia and lymphomas. However,an important limitation of allogeneic HCT is the development of graftversus host disease (GVHD), which occurs in a severe form in about30-50% of humans who receive this therapy.

Polypeptides of the invention are useful in preventing or reducingGraft-versus-Host-Disease (GvHD).

Accordingly, in one embodiment, the patient in need thereof is affectedwith a disease selected from the group consisting of acute myeloidleukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia(CML); myelodysplasia syndrome (MDS)/myeloproliferative syndrome;lymphomas such as Hodgkin and non-Hodgkin lymphomas, chronic lymphaticleukemia (CLL) and multiple myeloma.

In a third aspect, the invention relates to an isolated IL-34polypeptide for use in preventing or treating autoimmune diseases,unwanted immune responses against proteins expressed in the course ofgene therapy or therapeutic proteins and allergies in a patient thereof.

The invention also relates to an isolated M-CSF polypeptide for use inpreventing or treating autoimmune diseases, unwanted immune responsesagainst proteins expressed in the course of gene therapy or therapeuticproteins and allergies in a patient thereof.

The invention also relates to a combination of an isolated IL-34polypeptide and of an isolated M-CSF polypeptide for use in preventingor treating autoimmune diseases, unwanted immune responses againstproteins expressed in the course of gene therapy or therapeutic proteinsand allergies in a patient thereof.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the administration of therapy to an individual who may ultimatelymanifest at least one symptom of a disease, disorder, or condition, butwho has not yet done so, to reduce the chance that the individual willdevelop the symptom of the disease, disorder, or condition over a givenperiod of time. Such a reduction may be reflected, for example, in adelayed onset of the at least one symptom of the disease, disorder, orcondition in the patient.

As used herein, the terms “treat”, “treating” or “treatment” refers tothe administration of therapy to an individual in an attempt to reducethe frequency and/or severity of symptoms of a disease, defect,disorder, or adverse condition of a patient.

As used herein, the term “autoimmune disease” refers to a disease inwhich the immune system produces an immune response (for example, aB-cell or a T-cell response) against an antigen that is part of thenormal host (that is an auto-antigen), with consequent injury totissues. In an autoimmune disease, the immune system of the host failsto recognize a particular antigen as “self” and an immune reaction ismounted against the host's tissues expressing the antigen.

Exemplary autoimmune diseases affecting humans include rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(for example, Crohn's disease and ulcerative colitis), autoimmunegastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1diabetes, non-obese diabetes, myasthenia gravis, Grave's disease,Hashimoto's thyroiditis, sclerosing cholangitis, sclerosingsialadenitis, systemic lupus erythematosis, autoimmune thrombocytopeniapurpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis,polymyositis, dermatomyositis, acquired hemophilia, thromboticthrombocytopenic purpura and the like.

As used herein, the term “unwanted immune response against a therapeuticprotein” refers to any unwanted immune reaction directed to proteinsexpressed in the course of gene therapy, and/or therapeutic proteins,such as factor VIII (hemophilia A) and other coagulation factors, enzymereplacement therapies, monoclonal antibodies (e.g. natalizumab,rituximab, infliximab), polyclonal antibodies, enzymes or cytokines(e.g. IFN β).

For instance, this approach can indeed be applied to suppress an immuneresponse, especially to prevent immune reactions to specific proteinswhen their expression is restored by gene therapy in individuals withcorresponding genetic deficiencies. Thus, an isolated IL-34 polypeptideaccording to the invention may be used to prevent immune reactivitytowards proteins normally absent in the patient due to mutations, whiletheir reconstitution is achieved by gene therapy.

Moreover, protein therapy is an area of medical innovation that isbecoming more widespread, and involves the application of proteins, suchas enzymes, antibodies or cytokines, directly to patients as therapeuticproducts. One of the major hurdles in delivery of such medicamentsinvolves the immune responses directed against the therapeutic proteinthemselves. Administration of protein-based therapeutics is oftenaccompanied by administration of immune suppressants, which are used inorder to facilitate a longer lifetime of the protein and thereforeincreased uptake of the protein into the cells and tissues of theorganism. General immune suppressants can however be disadvantageous dueto the unspecific nature of the immune suppression that is carried out,resulting in unwanted side effects in the patient. Therefore, thisapproach can be applied to suppress an immune response againsttherapeutic proteins and peptides, such as therapeutic antibodies,cytokines, enzymes or any other protein administered to a patient.

As used herein, the term “allergy” or “allergies” refers to a disorder(or improper reaction) of the immune system. Allergic reactions occur tonormally harmless environmental substances known as allergens; thesereactions are acquired, predictable, and rapid. Strictly, allergy is oneof four forms of hypersensitivity and is called type I (or immediate)hypersensitivity. It is characterized by excessive activation of certainwhite blood cells called mast cells and basophils by a type of antibodyknown as IgE, resulting in an extreme inflammatory response. Commonallergic reactions include eczema, hives, hay fever, asthma, foodallergies, and reactions to the venom of stinging insects such as waspsand bees.

As used herein, the terms “Interleukin-34 polypeptide” or “IL-34polypeptide” are well known in the art and refer to a cytokine thatpromotes the proliferation, survival and differentiation of monocytesand macrophages. The term includes naturally occurring IL-34 isoforms(e.g. Q6ZMJ4 and Q6ZMJ4-2 with and without a Q81), variants (e.g.variants rs8046424 and rs7206509) and modified forms thereof. Thenaturally occurring human IL-34 protein has an aminoacid sequence of 242amino acids provided in the UniProt database under accession numberQ6ZMJ4 and is shown as follows (SEQ ID NO: 1) or a polypeptide having asequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical tothe sequence SEQ ID NO: 1:

MPRGFTWLRYLGIFLGVALGNEPLEMWPLTQNEECTVTGFLRDKLQYRSRLQYMKHYFPINYKISVPYEGVFRIANVTRLQRAQVSERELRYLWVLVSLSATESVQDVLLEGHPSWKYLQEVETLLLNVQQGLTDVEVSPKVESVLSLLNAPGPNLKLVRPKALLDNCFRVMELLYCSCCKQSSVLNWQDCEVPSPQSCSPEPSLQYAATQLYPPPPWSPSSPPHSTGSVRPVRAQGEGLLP

As used herein, the terms “macrophage colony stimulating factorpolypeptide” or “M-CSF polypeptide” (also known as CSF-1, for “colonystimulating factor 1 polypeptide”) refer to any native or variant(whether native or synthetic) cytokine which controls the production,differentiation, and function of macrophages. The term includesnaturally occurring M-CSF variants and modified forms thereof. Thus,three distinct variant M-CSF isoforms produced through alternative mRNAsplicing have been described, respectively a M-CSF[alpha] variant whichrefers to a protein of 256 amino acids provided in the UniProt Uniparcdatabase under accession number UPI0000D61F83, a M-CSF[beta] variantwhich refers to a protein of 554 amino acids provided in the GenPeptdatabase under accession number NP_000748.3 and is encoded by thenucleic acid sequence provided in the GenBank database under accessionnumber NM_000757.5, and a M-CSF[gamma] variant which refers to a proteinof 438 amino acids provided in the GenPept database under accessionnumber NP_757349.1 and is encoded by the nucleic acid sequence providedin the GenBank database under accession number NM 172210.2.

In one embodiment, the M-CSF polypeptide is the human isoformM-CSF[alpha] of 256 amino acids provided in the UniProt/Uniparc databaseunder accession number UPI0000D61F83 and is shown as follows (SEQ ID NO:4) or a polypeptide having a sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequence SEQ ID NO: 4:

MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQGHERQSEGSFSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDR QVELPV

In one embodiment, the M-CSF polypeptide is the human isoformM-CSF[beta] of 554 amino acids provided in the GenBank database underaccession number NP_000748.3 and is shown as follows (SEQ ID NO: 5) or apolypeptide having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequence SEQ ID NO: 5:

MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDVVTKPDCNCLYPKAIPSSDPASVSPHQPLAPSMAPVAGLTWEDSEGTEGSSLLPGEQPLHTVDPGSAKQRPPRSTCQSFEPPETPVVKDSTIGGSPQPRPSVGAFNPGMEDILDSAMGTNWVPEEASGEASEIPVPQGTELSPSRPGGGSMQTEPARPSNFLSASSPLPASAKGQQPADVTGTALPRVGPVRPTGQDWNHTPQKTDHPSALLRDPPEPGSPRISSLRPQGLSNPSTLSAQPQLSRSHSSGSVLPLGELEGRRSTRDRRSPAEPEGGPASEGAARPLPRFNSVPLTDTGHERQSEGSSSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDRQV ELPV

In one embodiment, the M-CSF polypeptide is the human isoformM-CSF[gamma] of 438 amino acids provided in the GenBank database underaccession number NP_757349.1_and is shown as follows (SEQ ID NO: 6) or apolypeptide having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequence SEQ ID NO: 6:

MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDVVTKPDCNCLYPKAIPSSDPASVSPHQPLAPSMAPVAGLTWEDSEGTEGSSLLPGEQPLHTVDPGSAKQRPPRSTCQSFEPPETPVVKDSTIGGSPQPRPSVGAFNPGMEDILDSAMGTNWVPEEASGEASEIPVPQGTELSPSRPGGGSMQTEPARPSNFLSASSPLPASAKGQQPADVTGHERQSEGSSSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDRQVELPV

As used herein, the term “polypeptide” refers to a polymer of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically, having no specific length. Thus, peptides, oligopeptidesand proteins are included in the definition of polypeptide and theseterms are used interchangeably throughout the specification, as well asin the claims. The term polypeptide does not exclude post-translationalmodifications that include but are not limited to phosphorylation,acetylation, glycosylation and the like. The term also applies to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

By an “isolated” polypeptide, it is intended that the polypeptide is notpresent within a living organism, e.g. within human body. However, theisolated polypeptide may be part of a composition or a kit. The isolatedpolypeptide is preferably purified. Such polypeptide is essentially freefrom contaminating cellular components, such as carbohydrate, lipid, orother proteinaceous impurities associated with the polypeptide innature. Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band followingSDS-polyacrylamide gel electrophoresis of the protein preparation andCoomassie Brilliant Blue staining of the gel.

The term “IL-34 polypeptide” is herein defined as including thenaturally occurring human polypeptide IL-34 and naturally-occurringallelic variations of the polypeptide. Allelic variations arenaturally-occurring base changes in the species population which may ormay not result in an amino acid change in a polypeptide or protein.Additionally, the IL-34 polypeptides according to the invention not onlyencompass polypeptides comprising or consisting of full-length IL-34 andvariants thereof, but also polypeptides consisting of fragments thereof,provided the fragments are biologically active. Additionally included inthis definition are both recombinant and synthetic versions of thepolypeptide IL-34, which may contain induced modifications in thepolypeptide and DNA sequences thereof. Accordingly, the term IL-34polypeptide intends to encompass the functional equivalents of the IL-34polypeptide encoded by the sequence SEQ ID NO: 1.

The term “M-CSF polypeptide” is herein defined as including thenaturally occurring human polypeptide M-CSF and naturally-occurringallelic variations of the polypeptide. Allelic variations arenaturally-occurring base changes in the species population which may ormay not result in an amino acid change in a polypeptide or protein.Additionally, the M-CSF polypeptides according to the invention not onlyencompass polypeptides comprising or consisting of full-length M-CSF andvariants thereof, but also polypeptides consisting of fragments thereof,provided the fragments are biologically active. Additionally included inthis definition are both recombinant and synthetic versions of thepolypeptide M-CSF, which may contain induced modifications in thepolypeptide and DNA sequences thereof. Accordingly, the term M-CSFpolypeptide intends to encompass the functional equivalents of the M-CSFpolypeptides encoded by the sequence SEQ ID NO: 4, SEQ ID NO: 5 or SEQID NO: 6.

As used herein, a “functional equivalent” refers to a molecule (e.g. arecombinant polypeptide) that retains the biological activity and thespecificity of the parent polypeptide. Therefore, the term “functionalequivalent of the IL-34 polypeptide” includes variants and fragments ofthe polypeptide to which it refers (i.e. the IL-34 polypeptide) providedthat the functional equivalents exhibit at least one, preferably all, ofthe biological activities of the reference polypeptide, as describedbelow. Functional equivalents of the IL-34 polypeptide have beenpreviously described (40). The term “functional equivalent of the M-CSFpolypeptide” includes variants and fragments of the polypeptide to whichit refers (i.e. the M-CSF polypeptide) provided that the functionalequivalents exhibit at least one, preferably all, of the biologicalactivities of the reference polypeptide, as described below.

A polypeptide “variant” refers to a biologically active polypeptidehaving at least about 80% amino acid sequence identity with the nativesequence polypeptide. Such variants include, for instance, polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the polypeptide. Ordinarily, a variant will have atleast about 80% amino acid sequence identity, more preferably at leastabout 90% amino acid sequence identity, and even more preferably atleast about 95% amino acid sequence identity with the native sequencepolypeptide.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid.

In the frame of the application, the percentage of identity iscalculated using a global alignment (i.e., the two sequences arecompared over their entire length). Methods for comparing the identityand homology of two or more sequences are well known in the art. The“needle” program, which uses the Needleman-Wunsch global alignmentalgorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to findthe optimum alignment (including gaps) of two sequences when consideringtheir entire length, may for example be used. The needle program is forexample available on the ebi.ac.uk world wide web site. The percentageof identity in accordance with the invention is preferably calculatedusing the EMBOSS::needle (global) program with a “Gap Open” parameterequal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62matrix.

Polypeptides consisting of an amino acid sequence “at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical” to a reference sequence maycomprise mutations such as deletions, insertions and/or substitutionscompared to the reference sequence. The polypeptide consisting of anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a reference sequence may correspond to an allelic variantof the reference sequence. It may for example only comprisesubstitutions compared to the reference sequence. The substitutionspreferably correspond to conservative substitutions as indicated in thetable below.

Conservative substitutions Type of Amino Acid Ala, Val, Leu, Ile, Aminoacids with aliphatic hydrophobic side chains Met, Pro, Phe, Trp Ser,Tyr, Asn, Gln, Amino acids with uncharged but polar side chains Cys Asp,Glu Amino acids with acidic side chains Lys, Arg, His Amino acids withbasic side chains Gly Neutral side chain

As used herein polypeptide, a “fragment” refers to a biologically activepolypeptide that is shorter than a reference polypeptide (i.e. afragment of the IL-34 polypeptide or a fragment of the M-CSFpolypeptide). Thus, the polypeptide according to the inventionencompasses polypeptides comprising or consisting of fragments of IL-34or M-CSF, provided the fragments are biologically active.

In the frame of the invention, the biologically active fragment may forexample comprise at least 175, 200, 205, 210, 215, 220, 225, 230, 235,240 consecutive amino acids of the IL-34 polypeptide.

In the frame of the invention, the biologically active fragment may forexample comprise at least 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525 or 550 consecutive amino acids of theM-CSF polypeptide.

In one particular embodiment, the M-CSF polypeptide comprises orconsists of a 150 amino acid polypeptide of human M-CSF and is shown asfollows (SEQ ID NO: 7):

MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVK

By “biological activity” of IL-34 or a functional equivalent thereof orM-CSF or a functional equivalent thereof is meant:

(i) the capacity to induce and/or maintain (at least 120 days in rat)immune tolerance (as described in the Section Examples; i.e. thecapacity to inhibit the CD4⁺ and CD8+ T cell proliferation in a mixedlymphocyte reaction (MLR); and/or

(ii) the capacity to prevent the transplant rejection in a model oforgan allotransplantation (model of cardiac allotransplantation).

The skilled in the art can easily determine whether a functionalequivalent of the IL-34 polypeptide or a functional equivalent of theM-CSF polypeptide is biologically active. To check whether the newlygenerated polypeptides inhibit the CD4⁺ T cell proliferation in a MLRand/or prevent the transplant rejection in a model of organallotransplantation, a FACS analysis or a single cell gene expressionprofiling (see in Example section) may be performed for eachpolypeptide. Moreover, to check whether the newly generated polypeptidesprevent the transplant rejection, a model of organ allotransplantationmay be used (see in Example section). Additionally, a time-course and adose-response performed in vitro or in vivo (e.g. by using a model oforgan allotransplantation) will determine the optimal conditions foreach polypeptide.

In one embodiment, the polypeptides of the invention may comprise a tag.A tag is an epitope-containing sequence which can be useful for thepurification of the polypeptides. It is attached to by a variety oftechniques such as affinity chromatography, for the localization of saidpolypeptide within a cell or a tissue sample using immuno labelingtechniques, the detection of said polypeptide by immunoblotting etc.Examples of tags commonly employed in the art are the GST(glutathion-S-transferase)-tag, the FLAG™-tag, the Strep-tag™, V5 tag,myc tag, His tag (which typically consists of six histidine residues),etc.

In another embodiment, the polypeptides of the invention may comprisechemical modifications improving their stability and/or theirbiodisponibility. Such chemical modifications aim at obtainingpolypeptides with increased protection of the polypeptides againstenzymatic degradation in vivo, and/or increased capacity to crossmembrane barriers, thus increasing its half-life and maintaining orimproving its biological activity. Any chemical modification known inthe art can be employed according to the present invention. Suchchemical modifications include but are not limited to:

-   -   replacement(s) of an amino acid with a modified and/or unusual        amino acid, e.g. a replacement of an amino acid with an unusual        amino acid like Nle, Nva or Orn; and/or    -   modifications to the N-terminal and/or C-terminal ends of the        peptides such as e.g. N-terminal acylation (preferably        acetylation) or desamination, or modification of the C-terminal        carboxyl group into an amide or an alcohol group;    -   modifications at the amide bond between two amino acids:        acylation (preferably acetylation) or alkylation (preferably        methylation) at the nitrogen atom or the alpha carbon of the        amide bond linking two amino acids;    -   modifications at the alpha carbon of the amide bond linking two        amino acids such as e.g. acylation (preferably acetylation) or        alkylation (preferably methylation) at the alpha carbon of the        amide bond linking two amino acids.    -   chirality changes such as e.g. replacement of one or more        naturally occurring amino acids (L enantiomer) with the        corresponding D-enantiomers;    -   retro-inversions in which one or more naturally-occurring amino        acids (L-enantiomer) are replaced with the corresponding        D-enantiomers, together with an inversion of the amino acid        chain (from the C-terminal end to the N-terminal end);    -   azapeptides, in which one or more alpha carbons are replaced        with nitrogen atoms; and/or    -   betapeptides, in which the amino group of one or more amino acid        is bonded to the β carbon rather than the α carbon.

Another strategy for improving drug viability is the utilization ofwater-soluble polymers. Various water-soluble polymers have been shownto modify biodistribution, improve the mode of cellular uptake, changethe permeability through physiological barriers; and modify the rate ofclearance from the body. To achieve either a targeting orsustained-release effect, water-soluble polymers have been synthesizedthat contain drug moieties as terminal groups, as part of the backbone,or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, givenits high degree of biocompatibility and ease of modification. Attachmentto various drugs, proteins, and liposomes has been shown to improveresidence time and decrease toxicity. PEG can be coupled to activeagents through the hydroxyl groups at the ends of the chain and viaother chemical methods; however, PEG itself is limited to at most twoactive agents per molecule. In a different approach, copolymers of PEGand amino acids were explored as novel biomaterials which would retainthe biocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule (providing greaterdrug loading), and which could be synthetically designed to suit avariety of applications.

Those of skill in the art are aware of PEGylation techniques for theeffective modification of drugs. For example, drug delivery polymersthat consist of alternating polymers of PEG and tri-functional monomerssuch as lysine have been used by VectraMed (Plainsboro, N.J.). The PEGchains (typically 2000 daltons or less) are linked to the a- and e-aminogroups of lysine through stable urethane linkages. Such copolymersretain the desirable properties of PEG, while providing reactive pendentgroups (the carboxylic acid groups of lysine) at strictly controlled andpredetermined intervals along the polymer chain. The reactive pendentgroups can be used for derivatization, cross-linking, or conjugationwith other molecules. These polymers are useful in producing stable,long-circulating pro-drugs by varying the molecular weight of thepolymer, the molecular weight of the PEG segments, and the cleavablelinkage between the drug and the polymer. The molecular weight of thePEG segments affects the spacing of the drug/linking group complex andthe amount of drug per molecular weight of conjugate (smaller PEGsegments provides greater drug loading). In general, increasing theoverall molecular weight of the block co-polymer conjugate will increasethe circulatory half-life of the conjugate. Nevertheless, the conjugatemust either be readily degradable or have a molecular weight below thethreshold-limiting glomular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintainingcirculatory half-life, and biodistribution, linkers may be used tomaintain the therapeutic agent in a pro-drug form until released fromthe backbone polymer by a specific trigger, typically enzyme activity inthe targeted tissue. For example, this type of tissue activated drugdelivery is particularly useful where delivery to a specific site ofbiodistribution is required and the therapeutic agent is released at ornear the site of pathology. Linking group libraries for use in activateddrug delivery are known to those of skill in the art and may be based onenzyme kinetics, prevalence of active enzyme, and cleavage specificityof the selected disease-specific enzymes. Such linkers may be used inmodifying the polypeptides described herein for therapeutic delivery.

In still another embodiment, the polypeptides of the invention may befused to a heterologous polypeptide (i.e. polypeptide derived from anunrelated protein, for example, from an immunoglobulin protein).

As used herein, the terms “fused” and “fusion” are used interchangeably.These terms refer to the joining together of two more elements orcomponents, by whatever means including chemical conjugation orrecombinant means. An “in-frame fusion” refers to the joining of two ormore polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. For instance, a recombinant fusion proteinmay be a single protein containing two or more segments that correspondto polypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence.

As used herein, the term “IL-34 fusion protein” refers to a polypeptidecomprising the IL-34 polypeptide or a functional equivalent thereoffused to heterologous polypeptide. The IL-34 fusion protein willgenerally share at least one biological property in common with theIL-34 polypeptide (as described above).

An example of an IL-34 fusion protein is an IL-34 immunoadhesin.

As used herein, the term “M-CSF fusion protein” refers to a polypeptidecomprising the M-CSF polypeptide or a functional equivalent thereoffused to heterologous polypeptide. The M-CSF fusion protein willgenerally share at least one biological property in common with theM-CSF polypeptide (as described above).

An example of a M-CSF fusion protein is a M-CSF immunoadhesin.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The immunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain (Fc region). Immunoadhesins can possessmany of the valuable chemical and biological properties of humanantibodies. Since immunoadhesins can be constructed from a human proteinsequence with a desired specificity linked to an appropriate humanimmunoglobulin hinge and constant domain (Fc) sequence, the bindingspecificity of interest can be achieved using entirely human components.Such immunoadhesins are minimally immunogenic to the patient, and aresafe for chronic or repeated use. In one embodiment, the Fc region is anative sequence Fc region. In another embodiment, the Fc region is avariant Fc region. In still another embodiment, the Fc region is afunctional Fc region. The IL-34 sequence portion and the immunoglobulinsequence portion of the IL-34 immunoadhesin may be linked by a minimallinker. The immunoglobulin sequence preferably, but not necessarily, isan immunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG1, IgG2, IgG3or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG1 or IgG3.

As used herein, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain, including native sequence Fcregions and variant Fc regions. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof.

Another example of an IL-34 fusion protein or M-CSF fusion protein is afusion of the IL-34 polypeptide or of the M-CSF polypeptide with humanserum albumin-binding domain antibodies (AlbudAbs) according to theAlbudAb™ Technology Platform as described in Konterman et al. 2012AlbudAb™ Technology Platform-Versatile Albumin Binding Domains for theDevelopment of Therapeutics with Tunable Half-Lives.

In another embodiment, the polypeptides of the invention may becombined/formulated with a drug delivery system suitable for therapeuticproteins.

Examples of such drug delivery system that may be used include variousmicro- as well as nanocarriers like microspheres/microparticles,liposomes, nanoparticles, dendrimers, niosomes and carbon nanotubes fortargeted delivery of therapeutic proteins. In a particular embodiment,such drug delivery system is a drug delivery system suitable forcell-mediated drug delivery, in particular monocytes- and/ormacrophages-mediated drug delivery as disclosed in Jain et al., 2013(42).

Alternatively, the polypeptides of the invention may be encapsulated inred blood cells or erythrocytes. Various methods have been described toallow the incorporation of active ingredients into red blood cellsincluding the method described in application EP 1 773 452 which is themethod currently offering the best performance and has the advantage ofbeing reproducible and of improving the encapsulation yield of activeingredient.

The polypeptides of the invention may be produced by any suitable means,as will be apparent to those of skill in the art. In order to producesufficient amounts of IL-34 or M-CSF polypeptides for use in accordancewith the invention, expression may conveniently be achieved by culturingunder appropriate conditions recombinant host cells containing thepolypeptide of the invention. Preferably, the polypeptide is produced byrecombinant means, by expression from an encoding nucleic acid molecule.Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known.

When expressed in recombinant form, the polypeptide is preferablygenerated by expression from an encoding nucleic acid in a host cell.Any host cell may be used, depending upon the individual requirements ofa particular system. Suitable host cells include bacteria mammaliancells, plant cells, yeast and baculovirus systems. Mammalian cell linesavailable in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells. HeLa cells, baby hamster kidneycells and many others. Bacteria are also preferred hosts for theproduction of recombinant protein, due to the ease with which bacteriamay be manipulated and grown. A common, preferred bacterial host is Ecoli.

Moreover, it should be noted that the majority of protein-basedbiopharmaceuticals bare some form of post-translational modificationwhich can profoundly affect protein properties relevant to theirtherapeutic application. Protein glycosylation represents the mostcommon modification (about 50% of human proteins are glycosylated).Glycosylation can introduce considerable heterogeneity into a proteincomposition through the generation of different glycan structures on theproteins within the composition. Such glycan structures are made by theaction of diverse enzymes of the glycosylation machinery as theglycoprotein transits the Endoplasmatic Reticulum (ER) and theGolgi-Complex (glycosylation cascade). The nature of the glycanstructure(s) of a protein has impact on the protein's folding,stability, life time, trafficking, pharmaco-dynamics, pharmacokineticsand immunogenicity. The glycan structure has great impact on theprotein's primary functional activity. Glycosylation can affect localprotein structure and may help to direct the folding of the polypeptidechain. One important kind of glycan structures are the so calledN-glycans. They are generated by covalent linkage of an oligosaccharideto the amino (N)-group of asparagin residues in the consensus sequenceNXS/T of the nascent polypeptide chain. N-glycans may furtherparticipate in the sorting or directing of a protein to its finaltarget: the N-glycan of an antibody, for example, may interact withcomplement components. N-glycans also serve to stabilize a glycoprotein,for example, by enhancing its solubility, shielding hydrophobic patcheson its surface, protecting from proteolysis, and directing intra-chainstabilizing interactions. Glycosylation may regulate protein half-life,for example, in humans the presence of terminal sialic acids inN-glycans may increase the half-life of proteins, circulating in theblood stream.

As used herein, the term “glycoprotein” refers to any protein having oneor more N-glycans attached thereto. Thus, the term refers both toproteins that are generally recognized in the art as a glycoprotein andto proteins which have been genetically engineered to contain one ormore N-linked glycosylation sites. As used herein, the terms “N-glycan”and “glycoform” are used interchangeably and refer to an N-linkedoligosaccharide, for example, one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-linked glycoproteins contain an N-acetylglucosamineresidue linked to the amide nitrogen of an asparagine residue in theprotein. The predominant sugars found on glycoproteins are glucose,galactose, mannose, fucose, N-acetylgalactosamine (GalNAc),N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminicacid (NANA)). The processing of the sugar groups occursco-translationally in the lumen of the ER and continuespost-translationally in the Golgi apparatus for N˜linked glycoproteins.

A number of yeasts, for example, Pichia pastoris, Yarrowia lipolyticaand Saccharomyces cerevisiae are recently under development to use theadvantages of such systems but to eliminate the disadvantages in respectto glycosylation. Several strains are under genetical development toproduce defined, human-like glycan structures on a protein. Methods forgenetically engineering yeast to produce human-like N-glycans aredescribed in U.S. Pat. Nos. 7,029,872 and 7,449,308 along with methodsdescribed in U.S. Published Application Nos. 20040230042, 20050208617,20040171826, 20050208617, and 20060286637. These methods have been usedto construct recombinant yeast that can produce therapeuticglycoproteins that have predominantly human-like complex or hybridN-glycans thereon instead of yeast type N-glycans. As previouslydescribed, human-like glycosylation is primarily characterized by“complex” N-glycan structures containing N-acetylglusosamine, galactose,fucose and/or N-acetylneuraminic acid. Thus, several strains of yeastshave been genetically engineered to produce glycoproteins comprising oneor more human-like complex or human-like hybrid N-glycans such asGlcNAcMan3GlcNAc2.

Alternatively, a nucleic acid encoding a polypeptide of the invention(such as the IL-34 polypeptide as shown in SEQ ID NO: 1 or the M-CSFpolypeptide as shown in SEQ ID NO: 4, 5, 6 and 7) or a vector comprisingsuch nucleic acid or a host cell comprising such vector may be used.

Accordingly, another aspect of the invention relates to a nucleic acidencoding an amino acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4,5, 6 and 7 as described here above, or a vector comprising such nucleicacid or a host cell comprising such vector, for use in inducing and/ormaintaining immune tolerance in a patient in need thereof.

Another aspect of the invention relates to a nucleic acid encoding anamino acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4, 5, 6 and 7as described here above, or a vector comprising such nucleic acid or ahost cell comprising such vector, for use in preventing or reducingtransplant rejection in a patient in need thereof.

Still another aspect of the invention relates to a nucleic acid encodingan amino acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4, 5, 6 and7 as described here above, or a vector comprising such nucleic acid or ahost cell comprising such vector, for use in preventing or treatingautoimmune diseases, alloimmune responses and allergies in a patientthereof.

Nucleic acids of the invention may be produced by any technique knownper se in the art, such as, without limitation, any chemical,biological, genetic or enzymatic technique, either alone or incombination(s).

In its broadest sense, a “vector” is any vehicle capable of facilitatingthe transfer of a nucleic acid to the cells. Preferably, the vectortransports the nucleic acid to cells with reduced degradation relativeto the extent of degradation that would result in the absence of thevector. In general, the vectors useful in the invention include, but arenot limited to, plasmids, phagemids, viruses, other vehicles derivedfrom viral or bacterial sources that have been manipulated by theinsertion or incorporation of the nucleic acid sequences of interest.Viral vectors are a preferred type of vector and include, but are notlimited to nucleic acid sequences from the following viruses:retrovirus, such as moloney murine leukemia virus, harvey murine sarcomavirus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in KRIEGLER (ALaboratory Manual,” W.H. Freeman C.O., New York, 1990) and in MURRY(“Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Cliffton,N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses andadeno-associated viruses, which are double-stranded DNA viruses thathave already been approved for human use in gene therapy. Theadeno-associated virus can be engineered to be replication deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages such as, heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See e.g., SANBROOK et al., “Molecular Cloning: A LaboratoryManual,” Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been used as DNA vaccines fordelivering antigen-encoding genes to cells in vivo. They areparticularly advantageous for this because they do not have the samesafety concerns as with many of the viral vectors. These plasmids,however, having a promoter compatible with the host cell, can express apeptide from a gene operatively encoded within the plasmid. Somecommonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, andpBlueScript. Other plasmids are well known to those of ordinary skill inthe art. Additionally, plasmids may be custom designed using restrictionenzymes and ligation reactions to remove and add specific fragments ofDNA. Plasmids may be delivered by a variety of parenteral, mucosal andtopical routes. For example, the DNA plasmid can be injected byintramuscular, intradermal, subcutaneous, or other routes. It may alsobe administered by intranasal sprays or drops, rectal suppository andorally. It may also be administered into the epidermis or a mucosalsurface using a gene-gun. The plasmids may be given in an aqueoussolution, dried onto gold particles or in association with another DNAdelivery system including but not limited to liposomes, dendrimers andmicroencapsulation.

According to the invention, examples of host cells that may be used arehuman monocytes or macrophages (particularly those obtained from thesubject to be treated).

The means by which the vector carrying the gene may be introduced intothe cells include, but are not limited to, microinjection,electroporation, transduction, or transfection using DEAE-dextran,lipofection, calcium phosphate or other procedures known to one skilledin the art.

Pharmaceutical Compositions

The invention relates to a pharmaceutical composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding thereof and anisolated M-CSF polypeptide or a polynucleotide encoding thereof.

The invention relates to a pharmaceutical composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding thereof and animmunosuppressive drug.

The invention relates to a pharmaceutical composition comprising anisolated M-CSF polypeptide or a polynucleotide encoding thereof and animmunosuppressive drug.

The invention also relates to a pharmaceutical composition comprising apolypeptide as defined herein (or a nucleic acid encoding therefor, anexpression vector or a host cell as above defined) and animmunosuppressive drug for use in inducing and/or maintaining immunetolerance in a patient in need thereof.

The invention further relates to a pharmaceutical composition comprisinga polypeptide as defined herein (or a nucleic acid encoding therefor, anexpression vector or a host cell as above defined) and animmunosuppressive drug for use in preventing or reducing transplantrejection in a patient in need thereof.

The invention relates to a pharmaceutical composition comprising apolypeptide as defined herein (or a nucleic acid encoding therefor, anexpression vector or a host cell as above defined) and animmunosuppressive drug for use in a patient in need thereof.

Pharmaceutical compositions comprising a polypeptide of the inventioninclude all compositions wherein the polypeptide is contained in anamount effective to achieve the intended purpose. In addition, thepharmaceutical compositions may contain suitable physiologicallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically.

The term “physiologically acceptable carrier” is meant to encompass anycarrier, which does not interfere with the effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which is administered. Suitable physiologically acceptablecarriers are well known in the art and are described for example inRemington's Pharmaceutical Sciences (Mack Publishing Company, Easton,USA, 1985), which is a standard reference text in this field. Forexample, for parenteral administration, the above active ingredients maybe formulated in unit dosage form for injection in vehicles such assaline, dextrose solution, serum albumin and Ringer's solution.

Besides the physiologically acceptable carrier, the pharmaceuticalcompositions of the invention can also comprise minor amounts ofadditives, such as stabilizers, excipients, buffers and preservatives.The pharmaceutical composition of the invention may further comprise animmunosuppressive drug.

In one embodiment, the immunosuppressive drug is selected from the groupconsisting of cytostatics such as mammalian target of rapamycin (mTOR)inhibitors and rapamycin (sirolimus); alkylating agents(cyclophosphamide) and antimetabolites (azathioprine, mercaptopurine andmethotrexate); therapeutic antibodies (such as anti-CD40L monoclonalantibodies, anti-IL-2R monoclonal antibodies, anti-CD3 monoclonalantibodies, anti-lymphocyte globulin (ALG) and anti-thymocyte globulin(ATG)); calcineurin inhibitors (cyclosporine); glucocorticoids andmycopheno late mofetil.

In one embodiment, the immunosuppressive drug is rapamycin (sirolimus).

The polypeptides of the invention may be administered by any means thatachieve the intended purpose. For example, administration may beachieved by a number of different routes including, but not limited tosubcutaneous, intravenous, intradermal, intramuscular, intraperitoneal,intracerebral, intrathecal, intranasal, oral, rectal, transdermal,buccal, topical, local, inhalant or subcutaneous use. Parenteral andtopical routes are particularly preferred.

Dosages to be administered depend on individual needs, on the desiredeffect and the chosen route of administration. It is understood that thedosage administered will be dependent upon the age, sex, health, andweight of the recipient, concurrent treatment, if any, frequency oftreatment, and the nature of the effect desired. The total dose requiredfor each treatment may be administered by multiple doses or in a singledose.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment. For example, it is well within the skillof the art to start doses of the compounds at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the polypeptides may be varied over a wide range from0.01 to 1,000 mg per adult per day. Preferably, the compositions contain0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250and 500 mg of the active ingredient for the symptomatic adjustment ofthe dosage to the subject to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferablyfrom 1 mg to about 100 mg of the active ingredient. An effective amountof the drug is ordinarily supplied at a dosage level from 0.0002 mg/kgto about 20 mg/kg of body weight per day, especially from about 0.001mg/kg to 10 mg/kg of body weight per day.

In one embodiment, the immunosuppressive drug is administered to thepatient in need thereof at a decreased dose (comparatively to the doseusually administered)

In one embodiment, the immunosuppressive drug is administered to thepatient in need thereof at a suboptimal dose.

In a particular embodiment, rapamycin (sirolimus) is administered to thepatient in need thereof at a suboptimal dose.

In one aspect, the invention relates to a method for inducing and/ormaintaining immune tolerance in a patient in need thereof, comprising astep of administering to said patient a therapeutically effective amountof an IL-34 polypeptide or a polynucleotide encoding therefor.

In another aspect, the invention relates to a method for inducing and/ormaintaining immune tolerance in a patient in need thereof, comprising astep of administering to said patient a therapeutically effective amountof a M-CSF polypeptide or a polynucleotide encoding therefor.

In another aspect, the invention relates to a method for inducing and/ormaintaining immune tolerance in a patient in need thereof, comprising astep of administering to said patient a therapeutically effective amountof a M-CSF polypeptide or a polynucleotide encoding therefor and atherapeutically effective amount of a IL-34 polypeptide or apolynucleotide encoding therefor.

In one embodiment, the method for inducing and/or maintaining immunetolerance in a patient in need thereof, comprising a step ofadministering to said patient a therapeutically effective amount of anIL-34 polypeptide or a polynucleotide encoding thereof and of atherapeutically effective amount an immunosuppressive agent.

In one embodiment, the method for inducing and/or maintaining immunetolerance in a patient in need thereof, comprising a step ofadministering to said patient a therapeutically effective amount of aM-CSF polypeptide or a polynucleotide encoding thereof and of atherapeutically effective amount an immunosuppressive agent.

In one embodiment, the method for inducing and/or maintaining immunetolerance in a patient in need thereof, comprising a step ofadministering to said patient a therapeutically effective amount of aM-CSF polypeptide or a polynucleotide encoding thereof, atherapeutically effective amount of a M-CSF polypeptide or apolynucleotide encoding thereof and of a therapeutically effectiveamount an immunosuppressive agent.

In another aspect, the invention relates to a method for preventing orreducing transplant rejection in a patient in need thereof, comprising astep of administering to said patient a therapeutically effective amountof an IL-34 polypeptide or a polynucleotide encoding thereforsimultaneously and/or subsequently to the transplantation.

In another aspect, the invention relates to a method for preventing orreducing transplant rejection in a patient in need thereof, comprising astep of administering to said patient a therapeutically effective amountof a M-CSF polypeptide or a polynucleotide encoding thereforsimultaneously and/or subsequently to the transplantation.

In another aspect, the invention relates to a method for preventing orreducing transplant rejection in a patient in need thereof, comprising astep of administering to said patient a therapeutically effective amountof a M-CSF polypeptide or a polynucleotide encoding therefor, atherapeutically effective amount of a IL-34 polypeptide or apolynucleotide encoding therefor simultaneously and/or subsequently tothe transplantation.

In one embodiment, the method for preventing or reducing transplantrejection in a patient in need thereof, comprising a step ofadministering to said patient a therapeutically effective amount of anIL-34 polypeptide or a polynucleotide encoding therefor and of atherapeutically effective amount an immunosuppressive agentsimultaneously and/or subsequently to the transplantation.

In one embodiment, the method for preventing or reducing transplantrejection in a patient in need thereof, comprising a step ofadministering to said patient a therapeutically effective amount of anM-CSF polypeptide or a polynucleotide encoding therefor and of atherapeutically effective amount an immunosuppressive agentsimultaneously and/or subsequently to the transplantation.

In one embodiment, the method for preventing or reducing transplantrejection in a patient in need thereof, comprising a step ofadministering to said patient a therapeutically effective amount of anM-CSF polypeptide or a polynucleotide encoding therefor, atherapeutically effective amount of a IL-34 polypeptide or apolynucleotide encoding therefor and a therapeutically effective amountan immunosuppressive agent simultaneously and/or subsequently to thetransplantation.

As used herein, the term “simultaneously” means that the polypeptide ofinterest is administered to the recipient patient the same day that thetransplantation.

As used herein, the term “subsequently” means that the polypeptide ofinterest is administered to the recipient patient after thetransplantation, for instance 2, 3, 4, 5, 6 or 7 days following thetransplantation.

In a further aspect, the invention relates to a method for improvingsurvival of a transplant, said method comprising a step of(pre-)culturing the transplant with a culture medium comprising aneffective amount of an IL-34 polypeptide or a polynucleotide encodingtherefor and/or a M-CSF polypeptide or a polynucleotide encodingtherefor.

As used herein, the term “culture medium” refers to a liquid mediumsuitable for the ex vivo culture of mammalian cells, tissues or organs.

The culture medium used by the invention may be a water-based mediumthat includes a combination of substances such as salts, nutrients,minerals, vitamins, amino acids, nucleic acids, proteins such ascytokines, growth factors and hormones, all of which are needed forcell, tissue or organ survival.

For example, a culture medium according to the invention may be asynthetic tissue culture medium such as the RPMI (Roswell Park MemorialInstitute medium) or the CMRL-1066 (Connaught Medical ResearchLaboratory) for human use, supplemented with the necessary additives.

In a preferred embodiment, the culture medium of the invention is freeof animal-derived substances. In a preferred embodiment, the culturemedium of the invention consists essentially of synthetic compounds,compounds of human origin and water. Advantageously, said culture mediumcan be used for culturing cells according to good manufacturingpractices (under “GMP” conditions).

In a further aspect, the invention relates to a method for improvingsurvival of the transplant in a transplanted patient (recipient), saidmethod comprising a step of administering a therapeutically effectiveamount of an IL-34 polypeptide or a polynucleotide encoding therefor tosaid patient and/or a M-CSF polypeptide or a polynucleotide encodingtherefor.

In one embodiment, the IL-34 polypeptide or a polynucleotide encodingtherefor and/or the M-CSF polypeptide or a polynucleotide encodingtherefor is administrated to the patient in the very first phase oftransplantation.

In still another aspect, the invention relates to a method forpreventing or treating autoimmune diseases, unwanted or anti-therapeuticproteins immune responses and allergies in a patient in need thereof,comprising a step of administering to said patient a therapeuticallyeffective amount of an IL-34 polypeptide or a polynucleotide encodingtherefor and/or a M-CSF polypeptide or a polynucleotide encodingtherefor.

In one embodiment, the method for preventing or treating autoimmunediseases, alloimmune responses and allergies in a patient in needthereof, comprising a step of administering to said patient atherapeutically effective amount of an IL-34 polypeptide or apolynucleotide encoding thereof and/or a M-CSF polypeptide or apolynucleotide encoding therefor and of a therapeutically effectiveamount an immunosuppressive agent.

By “therapeutically effective amount” is meant an amount sufficient toachieve a concentration of polypeptide which is capable of preventing,treating or slowing down the disease to be treated. Such concentrationscan be routinely determined by those of skilled in the art. The amountof the polypeptide actually administered will typically be determined bya physician or a veterinarian, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual compound administered, the age, weight,and response of the patient, the severity of the subject's symptoms, andthe like. It will also be appreciated by those of skilled in the artthat the dosage may be dependent on the stability of the administeredpolypeptide.

In one embodiment, the treatment with an IL-34 polypeptide and/or aM-CSF polypeptide is administered in more than one cycle, i.e. theadministration of an IL-34 polypeptide and/or a M-CSF polypeptide isrepeated at least once.

For example, 2 to 10 cycles or even more, depending on the specificpatient status and response, may be administered. The intervals, i.e.the time between the start of two subsequent cycles, are typicallyseveral days.

Kit-of-Part Compositions

The IL-34 polypeptide and the M-CSF polypeptide may be combined withinone formulation and administered simultaneously. The invention thusrelates to a kit-of-part composition comprising an isolated IL-34polypeptide or a polynucleotide encoding therefor and a M-CSFpolypeptide or a polynucleotide encoding therefor.

The IL-34 polypeptide and/or the M-CSF polypeptide and theimmunusuppressive drug may be combined within one formulation andadministered simultaneously. However, they may also be administeredseparately, using separate compositions. It is further noted that theymay be administered at different times.

The invention thus relates to a kit-of-part composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding therefor and/ora M-CSF polypeptide or a polynucleotide encoding therefor and animmunosuppressive drug.

The invention also relates to a kit-of-part composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding therefor and/ora M-CSF polypeptide or a polynucleotide encoding therefor and animmunosuppressive drug for use in inducing and/or maintaining immunetolerance in a patient in need thereof.

The invention also relates to a kit-of-part composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding therefor and/ora M-CSF polypeptide or a polynucleotide encoding therefor and animmunosuppressive drug for use in preventing or reducing transplantrejection in a patient in need thereof.

The invention further relates to a kit-of-part composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding therefor and/ora M-CSF polypeptide or a polynucleotide encoding therefor and animmunosuppressive drug for use in preventing or treating autoimmunediseases, alloimmune responses and allergies in a patient in needthereof.

The terms “kit”, “product” or “combined preparation”, as used herein,define especially a “kit-of-parts” in the sense that the combinationpartners as defined above can be dosed independently or by use ofdifferent fixed combinations with distinguished amounts of thecombination partners, i.e. simultaneously or at different time points.The parts of the kit of parts can then, e.g., be administeredsimultaneously or chronologically staggered, that is at different timepoints and with equal or different time intervals for any part of thekit of parts. The ratio of the total amounts of the combination partnersto be administered in the combined preparation can be varied. Thecombination partners can be administered by the same route or bydifferent routes. When the administration is sequential, the firstpartner may be for instance administered 1, 2, 3, 4, 5, 6, 7, daysbefore the second partner.

Prognostic Methods of the Invention

In a further aspect, the invention relates to an in vitro method fordetermining whether a patient is at risk of transplant rejectionautoimmune diseases, unwanted immune response against therapeuticproteins or allergies, comprising a step of determining the expressionlevel of IL-34 in a biological sample obtained from said patient,wherein the presence of IL-34 is indicative of a reduced risk oftransplant rejection autoimmune diseases, unwanted immune responseagainst therapeutic proteins or allergies.

In a further aspect, the invention relates to an in vitro method fordetermining whether a patient is at risk of transplant rejectionautoimmune diseases, unwanted immune response against therapeuticproteins or allergies, comprising a step of determining the expressionlevel of M-CSF in a biological sample obtained from said patient,wherein the presence of M-CSF is indicative of a reduced risk oftransplant rejection autoimmune diseases, unwanted immune responseagainst therapeutic proteins or allergies.

As used herein, the term “risk” refers to the probability that an eventwill occur over a specific time period, such as the onset of transplantrejection, and can mean a subject's “absolute” risk or “relative” risk.Absolute risk can be measured with reference to either actualobservation post-measurement for the relevant time cohort, or withreference to index values developed from statistically valid historicalcohorts that have been followed for the relevant time period. Relativerisk refers to the ratio of absolute risks of a patient compared eitherto the absolute risks of low risk cohorts or an average population risk,which can vary by how clinical risk factors are assessed. Odds ratios,the proportion of positive events to negative events for a given testresult, are also commonly used (odds are according to the formulap/(1−p) where p is the probability of event and (1−p) is the probabilityof no event) to no-conversion.

“Risk determination” in the context of the invention encompasses makinga prediction of the probability, odds, or likelihood that an event mayoccur. Risk determination can also comprise prediction of futureclinical parameters, traditional laboratory risk factor values, suchage, sex mismatch, HLA-testing, etc . . . ; either in absolute orrelative terms in reference to a previously measured population. Themethods of the invention may be used to make categorical measurements ofthe risk of transplant rejection, thus defining the risk spectrum of acategory of transplanted patient defined as being at risk of transplantrejection.

In a still further aspect, the invention relates to an in vitro methodfor determining whether a transplanted patient (recipient) is tolerant,comprising a step of determining the expression level of IL-34 in abiological sample obtained from said transplanted patient, wherein thepresence of IL-34 is indicative of tolerance.

In a still further aspect, the invention relates to an in vitro methodfor determining whether a transplanted patient (recipient) is tolerant,comprising a step of determining the expression level of M-CSF in abiological sample obtained from said transplanted patient, wherein thepresence of M-CSF is indicative of tolerance.

As used herein, the term “determining” includes qualitative and/orquantitative detection (i.e. detecting and/or measuring the expressionlevel) with or without reference to a control or a predetermined value.As used herein, “detecting” means determining if IL-34 or M-CSF ispresent or not in a biological sample and “measuring” means determiningthe amount of IL-34 or M-CSF in a biological sample. Typically theexpression level may be determined for example by immunoassays such asan ELISA performed on a biological sample.

As used herein, the term “biological sample” has its general meaning inthe art and refers to any sample which may be obtained from a patientfor the purpose of in vitro evaluation. A preferred biological sample isa blood sample (e.g. whole blood sample, serum sample, or plasmasample).

Methods for Determining the Expression Level of the Biomarker of theInvention:

Determination of the expression level of IL-34 or M-CSF may be performedby a variety of techniques. Generally, the expression level asdetermined is a relative expression level.

For example, the determination of the expression level of IL-34 or M-CSFmay comprise a step of contacting the biological sample with selectivereagents such as antibodies, and thereby detecting the presence, ormeasuring the amount, of polypeptide of interest originally in saidbiological sample. Contacting may be performed in any suitable device,such as a plate, microtiter dish, test tube, well, glass, column, and soforth.

In one embodiment, the contacting is performed on a substrate coatedwith the reagent. The substrate may be a solid or semi-solid substratesuch as any suitable support comprising glass, plastic, nylon, paper,metal, polymers and the like. The substrate may be of various forms andsizes, such as a slide, a membrane, a bead, a column, a gel, etc. Thecontacting may be made under any condition suitable for a detectablecomplex, such as an antibody-antigen complex, to be formed between thereagent and the polypeptides of the biological sample.

The presence of the IL-34 polypeptide or M-CSF polypeptide may bedetected using standard electrophoretic and immunodiagnostic techniques,including immunoassays such as competition, direct reaction, or sandwichtype assays. Such assays include, but are not limited to, Western blots;agglutination tests; enzyme-labelled and mediated immunoassays, such asELISAs; biotin/avidin type assays; radioimmunoassays;immunoelectrophoresis; immunoprecipitation, etc. The reactions generallyinclude revealing labels such as fluorescent, chemiluminescent,radioactive, enzymatic labels or dye molecules, or other methods fordetecting the formation of a complex between the antigen and theantibody or antibodies reacted therewith. Labels are known in the artthat generally provide (either directly or indirectly) a signal.

As used herein, the term “labelled” with regard to the antibody oraptamer, is intended to encompass direct labelling of the antibody oraptamer by coupling (i.e., physically linking) a detectable substance,such as a radioactive agent or a fluorophore (e.g. fluoresceinisothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5), to theantibody or aptamer, as well as indirect labelling of the probe orantibody (e.g., horseradish peroxidise, HRP) by reactivity with adetectable substance. An antibody or aptamer may be also labelled with aradioactive molecule by any method known in the art. For example,radioactive molecules include but are not limited radioactive atom forscintigraphic studies such as I123, I124, In111, Re186 and Re188. Theaforementioned assays generally involve separation of unbound protein ina liquid phase from a solid phase support to which antigen-antibodycomplexes are bound. Solid supports which may be used in the practice ofthe invention include substrates such as nitrocellulose (e.g., inmembrane or microtiter well form); polyvinylchloride (e.g., sheets ormicrotiter wells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, etc.

More particularly, an ELISA method may be used, wherein the wells of amicrotiter plate are coated with an antibody against the protein to betested. A biological sample containing or suspected of containing thebiomarker is then added to the coated wells. After a period ofincubation sufficient to allow the formation of antibody-antigencomplexes, the plate(s) can be washed to remove unbound moieties and adetectably labelled secondary binding molecule added. The secondarybinding molecule is allowed to react with any captured sample markerprotein, the plate washed and the presence of the secondary bindingmolecule detected using methods well known in the art.

The selective reagent is generally an antibody that may be polyclonal ormonoclonal, preferably monoclonal. Polyclonal antibodies directedagainst IL-34 are well known from the skilled man in the art such as theantibodies commercialized by Abnova PAB16574.

Monoclonal antibodies directed against IL-34 are also well known such asthe monoclonal antibody commercialized by Abnova MAB10698.

Additionally, IL-34 ELISA Kits are also well known such as thosecommercialized by Abnova KA2217 Kit or by R&D Systems (Human IL-34Quantikine ELISA).

The selective reagent is generally an antibody that may be polyclonal ormonoclonal, preferably monoclonal. Polyclonal antibodies directedagainst M-CSF are well known from the skilled man in the art such as theantibodies commercialized by Abcam (ab9693).

Monoclonal antibodies directed against M-CSF are also well known such asthe monoclonal antibody commercialized by MyBioSource (MBS690427).

Additionally, M-CSF ELISA Kits are also well known such as thosecommercialized by R&D Systems (Human M-CSF Quantikine ELISA).

In a particular embodiment, the expression level of IL-34 or M-CSF isdetermined by measuring the concentration of IL-34 or M-CSF in saidbiological sample.

Accordingly, the methods according to the invention comprise a step ofcontacting the blood sample with a binding partner capable ofselectively interacting with IL-34 polypeptide or M-CSF polypeptide insaid blood sample.

The binding partner may be generally an antibody that may be polyclonalor monoclonal, preferably monoclonal. Polyclonal antibodies directedagainst IL-34 or M-CSF can be raised according to known methods byadministering the appropriate antigen or epitope to a host animalselected, e.g., from pigs, cows, horses, rabbits, goats, sheep, andmice, among others. Various adjuvants known in the art can be used toenhance antibody production.

Monoclonal antibodies of the invention can be prepared and isolatedusing any technique that provides for the production of antibodymolecules by continuous cell lines in culture. Techniques for productionand isolation include but are not limited to the hybridoma techniqueoriginally described by Kohler and Milstein (1975); the human B-cellhybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique(Cole et al., 1985). Alternatively, techniques described for theproduction of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778)can be adapted to produce single chain antibodies. Antibodies useful inpracticing the present invention also include fragments including butnot limited to F(ab′)2 fragments, which can be generated by pepsindigestion of an intact antibody molecule, and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab and/or scFv expression libraries can be constructedto allow rapid identification of fragments having the desiredspecificity. For example, phage display of antibodies may be used. Insuch a method, single-chain Fv (scFv) or Fab fragments are expressed onthe surface of a suitable bacteriophage, e. g., M13. Briefly, spleencells of a suitable host, e. g., mouse, that has been immunized with aprotein are removed. The coding regions of the VL and VH chains areobtained from those cells that are producing the desired antibodyagainst the protein. These coding regions are then fused to a terminusof a phage sequence. Once the phage is inserted into a suitable carrier,e. g., bacteria, the phage displays the antibody fragment. Phage displayof antibodies may also be provided by combinatorial methods known tothose skilled in the art. Antibody fragments displayed by a phage maythen be used as part of an immunoassay.

In another embodiment, the binding partner may be an aptamer. Aptamersare a class of molecule that represents an alternative to antibodies interm of molecular recognition. Aptamers are oligonucleotide oroligopeptide sequences with the capacity to recognize virtually anyclass of target molecules with high affinity and specificity. Suchligands may be isolated through Systematic Evolution of Ligands byEXponential enrichment (SELEX) of a random sequence library, asdescribed in Tuerk C. and Gold L., 1990. The random sequence library isobtainable by combinatorial chemical synthesis of DNA. In this library,each member is a linear oligomer, eventually chemically modified, of aunique sequence. Possible modifications, uses and advantages of thisclass of molecules have been reviewed in Jayasena S.D., 1999. Peptideaptamers consist of conformationally constrained antibody variableregions displayed by a platform protein, such as E. coli Thioredoxin A,that are selected from combinatorial libraries by two hybrid methods(Colas et al., 1996).

In other embodiments, measuring the concentration of IL-34 or M-CSF mayalso include separation of the proteins: centrifugation based on theprotein's molecular weight; electrophoresis based on mass and charge;HPLC based on hydrophobicity; size exclusion chromatography based onsize; and solid-phase affinity based on the protein's affinity for theparticular solid-phase that is use. Once separated, IL-34 or M-CSF maybe identified based on the known “separation profile” e. g., retentiontime, for that protein and measured using standard techniques.Alternatively, the separated proteins may be detected and measured by,for example, a mass spectrometer.

Methods for Adjusting an Immunosuppressive Treatment

The invention further provides methods for developing personalizedtreatment plans. Information gained by way of the methods describedabove can be used to develop a personalized treatment plan for atransplant recipient.

Accordingly, in a further aspect, the invention relates to a method foradjusting the immunosuppressive treatment administered to a patient inneed thereof, comprising the following steps of (i) performing themethod for determining the risk according to the invention, and (ii)adjusting the immunosuppressive treatment.

The methods can be carried out by, for example, using any of the methodsfor determining risk described above and, in consideration of theresults obtained, designing a treatment plan for the transplantrecipient. If IL-34 or M-CSF is not present in the biological sampleobtained from a patient of interest, this indicates that said patient isat risk for an undesirable clinical outcome (e.g., transplantrejection). Therefore, said patient is a candidate for treatment with aneffective amount of an immunosuppressive treatment (e.g. by ananti-rejection agent). On the contrary, the presence of IL-34 or M-CSFin the biological sample is indicative of a reduced risk of transplantrejection. Moreover, depending on the expression level IL-34 or M-CSF(i.e. low level or high level of IL-34 or M-CSF in the analyzedbiological sample), the patient may require a treatment regime that ismore or less aggressive than a standard regimen, or it may be determinedthat the patient is best suited for a standard regimen. For instance, apatient with a high level of IL-34 or M-CSF may avoid animmunosuppressive treatment (or require a less aggressive regimen) andtheir associated side effects.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Increased IL-34 expression in grafts and splenic CD8+ Tregsfollowing treatment with CD40Ig. A. FACS Aria-sorted CD8+CD45RClow Tregsfrom spleen of naive or 120 days old AdCD40Ig-treated recipients (n=6)were analysed for IL-34 mRNA expression by quantitative RT-PCR. Cardiacgrafts (B) and spleen (C) from AdCD40Ig-treated recipients at day 5(n=3) and 120 (n=7) after transplantation was compared with grafts fromnon-treated-recipients at day 5 (n=8), day 7 (n=8) and day 120 (n=6) andnative hearts from naive animals (n=7) for IL-34 mRNA expression. (D)CD8⁺CD45RC^(low) Tregs were sorted from the spleen of CD40Ig-treatedrats and analyzed for their expression of IL-34 (black) after 7 hoursstimulation with PMA ionomycin (24 μg/mL). Grey-filled histogramrepresents the isotype control staining. Mann Whitney, *p<0.05, **p<0.01.

FIG. 2: IL-34 and CD115, but not M-CSF, were involved in CD8+CD45RClowTreg-mediated suppression. The relative proportion of CFSE-labeledLEW.1A dividing CD4+CD25− T cells stimulated with donor LEW.1W pDCs wasanalyzed after 6 days of culture, in the presence of LEW.1ACD8+CD45RClow Tregs at a 1:1 effector:suppressor ratio. Theproliferation after addition of an anti-IL-34-blocking Ab (A), ananti-M-CSF blocking Ab (B) or an anti-CD115 blocking Ab (C) wasevaluated compared to isotypic control (n=4 in triplicates). Results areexpressed as mean±SEM of normalized percentage of proliferation vs.proliferation in the absence of CD8+Tregs (100%).*, p<0.01.

FIG. 3: Detection of IL-34 using an AAV-IL-34 and inhibition of allo-Tcell responses. A. Supernatants of AAV-IL-34 or AAV-GFP-transduced cellswere tested for suppression of CD4⁺CD25⁻ T cell proliferation inresponse to allogeneic pDCs and analysed by flow cytometry for CFSEdilution after 5 days of culture. CD8⁺ Tregs were used as positivecontrol of suppression. n=3 in duplicates; Results are expressed asmean±SEM of normalized percentage of proliferation vs. proliferation inthe absence of CD8⁺ Tregs (100%). Representative histogram of 1experiment; filled grey: proliferation of CD4⁺ T cells cocultured withpDC with 20% AAVGFP-transduced cell supernatent; black line: with 20%AAV-IL-34-transduced cells supernatant. B. Serial dilution of sera fromAAV-IL-34 or AAV-GFP-treated rats or naive animals were tested forsuppression of CD4⁺CD25⁻ T cell proliferation in response to allogeneicpDCs and analysed by flow cytometry for CFSE dilution after 5 days ofculture. CD8⁺ Tregs were used as positive control of suppression. n=3 induplicates; Results are expressed as mean±SEM of normalized percentageof proliferation vs. proliferation in the absence of CD8⁺ Tregs (100%).*, p<0.01; **, p<0.001; ***, p<0.0001 vs. sera from naïve animals.

FIG. 4: IL-34 gene transfer prolongs allograft survival in a dominantmanner. Recipients received intravenously 10¹² vg/rat of AAV-IL-34 ornon-coding AAV or untreated, were grafted 30 days later with noadditional treatment or in combination with suboptimal dose of rapamycin(14 days, starting day0). Graft survival was evaluated by palpationthrough the abdominal wall. ***p<0.0001.

FIG. 5: Serial tolerance mediated by Tregs after IL-34 induction. A.LEW.1A recipients were sublethally irradiated (4.5 Gy) at day −1 andreceived heart allografts and i.v. injections of 1,5.10⁸ splenocytesfrom long surviving recipient or naive animals at day 0. Graft survivalwas monitored by abdominal palpation. B. LEW.1A recipients weresublethally irradiated (4.5 Gy) at day −1 and received heart allograftsand i.v. injections of total splenocytes or purified sub-populations (Tcells: 4.10⁷; B cells: 6.10⁷, CD11b/c+ cells: 1,5.10⁷; CD8⁺CD45RC^(low)Tregs: 4×10⁶; CD4⁺CD25^(high) Tregs: 4×10⁶) from long survivingrecipient at day 0. Graft survival was monitored by abdominal palpation.Log Rank test, ** p<0.01, *p<0.05.

FIG. 6: M-CSF mediated dose dependent suppression of anti-donor effectorCD4^(÷)CD25⁻ T cells responses. (A) FACS Aria-sorted CD8⁺CD45RC^(low)Tregs from spleen of naive or 120 days old AdCD40Ig-treated recipients(n=6) were analysed for M-CSF mRNA expression by quantitative RT-PCR.*p<0.05. (B) Rat M-CSF (0.1 to 2 μg/ml final concentration) was testedfor suppressive activity on CFSE-labelled CD4⁺CD25⁻ T-cell proliferationafter 6 days of culture. CD8⁺ Tregs were used as positive control ofsuppression. n=3 in triplicates. Results are expressed as mean±SEM ofnormalized percentage of proliferation vs. proliferation in the absenceof CD8⁺ Tregs (100%). *, p<0.01.

FIG. 7: Transcript accumulation in macrophages following IL34 treatment.mRNA expression was assessed by real-time quantitative PCR on sortedmacrophages from untreated spleen or AAV-IL34-treated spleen, blood andgraft of recipients at day 15 post-transplantation. Results arenormalized to HPRT and expressed as 2^(−ddcT)+/−SEM. Kruskal Wallis andDunn's post test, *, p<0.01.

FIG. 8: Macrophages depletion resulted in allograft rejection followingIL34 treatment. Recipients receiving at day −30 intravenously 10¹²vg/rat of AAV-IL34 or non-coding AAV or were untreated with or withoutweekly intraperitoneal administration of clodronate liposomes from day−25 to day 3, and were grafted at day 0 in combination with suboptimaldose of rapamycin (10 days, starting day 0). Graft survival wasevaluated by palpation through the abdominal wall. Log Rank test,**p<0.01.

FIG. 9: IL34 is involved in human CD4+ and CD8+ Treg's suppressiveactivity, that can inhibit anti-donor immune responses. (A) Soluble IL34was tested for suppression of CD4⁺CD25⁻ T cell proliferation in responseto allogeneic T-depleted PBMCs and analyzed by flow cytometry for CFSEdilution after 5 days of culture. n=2 to 5 in duplicates; Results areexpressed as mean±SEM of relative proportion of dividing CD4⁺CD25⁻ Tcells. (B) The relative proportion of CFSE-labeled dividing CD4⁺CD25⁻ Tcells stimulated with allogeneic T-depleted PBMCs was analyzed after 5days of culture, in the presence of CD8⁺CD45RC^(low) or CD4⁺CD25^(high)Tregs at 1:1 effector:suppressor ratios. The proliferation afteraddition of anti-IL34-blocking Ab was evaluated and compared to isotypiccontrol (n=5-6 in triplicates). The proportion of dividing CD4⁺CD25⁻ Tcells in the control proliferation condition with allogeneic T-depletedPBMCs only represented approximately 60% of the cells on day 5 and wasgiven a value of 100 in each experiment. Results are expressed asmean±SEM of the relative proportion of dividing CD4⁺CD25⁻ T cells.

EXAMPLE 1: INTERLEUKIN-34, A NEW TREG-SPECIFIC CYTOKINE MEDIATOR OFTRANSPLANT TOLERANCE

Material & Methods

Healthy Volunteer Blood Collection and PBMC Separation:

Blood was collected from healthy donors, after informed consent wasgiven, at the Etablissement Francais du Sang (Nantes, France). Blood wasdiluted 2-fold with PBS before PBMCs were isolated by Ficoll-Paquedensity-gradient centrifugation (Eurobio) at 2000 rpm for 30 min at roomtemperature without braking. Collected PBMCs were washed in 50 mL PBS at1800 rpm for 10 min.

Animals and Cardiac Transplantation Models:

Heart allotransplantation was performed between whole MHC incompatiblemale LEW-1W (donors) and LEW-1A (recipients) rats as previouslydescribed (15). Heart survival was evaluated by palpation through theabdominal wall and heart beating was graded from +++ to −. Theexperiments were approved by the regional ethical committee for animalexperimentation.

IL-34 Quantitative RT-PCR:

The isolation and retrotranscription of mRNA as well as thequantification of specific mRNA levels using Taqman technology afternormalization to HPRT values have been described (15). The probessequences were for forward primer 5′ CTGGCTGTCCTCTACCCTGA 3′ (SEQ ID NO:2) and for reverse primer 5′ TGTCGTGGCAAGATATGGCAA 3′ (SEQ ID NO: 3).

Cell Sorting, Monoclonal Antibodies and Flow Cytometry:

Macrophages were sorted on TCRαβ (R7/3) and TCRγδ (V65) negative cells,CD45RA-FITC (OX33), and CD11b/c-APC (OX42) positive cells. Naive LEW.1ACD4⁺CD25⁻ T cells, LEW.1W pDC and LEW.1A CD8⁺CD45RC^(low) Tregs subsetswere sorted as previously described (16). The antibodies used for Tcells (TCRαβ, clone R7/3), CD4⁺CD25⁻ T cells (clones OX35 and OX39),CD8⁺ T cells (clone OX8), CD8⁺CD45RC^(low) T cells (clones OX8 andOX22), and CD4⁺CD45R⁺85C7⁺ pDCs (clones His24, OX35 and 85C7) sortingwere obtained from the European Collection of Cell Culture (Salisbury,UK). All biotin-labelled mAbs were visualized using Strepavidin-PE-Cy7(BD Biosciences) or Streptavidin-Alexa405. Human CD4⁺CD25⁻ T cells weresorted by gating on CD3⁺CD4⁺CD25⁻ cells (clones SKY7, L-200 and MA251),CD4⁺ Tregs by gating on CD25high and CD127low cells (clone HIL7-R M21),and CD8⁺ Tregs by gating on CD3+CD4-CD45RClow cells (clone MT2).IL-34-Myc was detected using an anti-myc antibody (9E10, Sigma). IL-34,CD115, and MCSF were blocked with the anti-IL-34 (PAB16574, Abnova),anti-CD115 (MCA1898, Serotec) and anti-M-CSF (AB-416-NA, R and D system)antibodies. Antibodies against MHC-II (OX6), CD11b and CD45RA⁺ B cells(OX33) were analysed to characterize cell's phenotype. Antibodiesagainst CD3-PeCy7 (SKY7), CD4-PercPCy5.5 (L200), CD25-APCCy7 (M-A251),CD127-PE (HIL7-R M21, BD Bioscience), CD45RC-FITC (MT2, IQ Product),Foxp3-APC (236A/E7, ebiosciences) and IL34-PE (578416, R&D) were used tocharacterize human cell phenotypes. A FACS ARIA I (BD Biosciences,Mountain View, Calif.) was used to sort cells. A Canto II cytometer (BDBiosciences, Mountain View, Calif.) was used to measure fluorescence,and data were analyzed using the FLOWJO software (Tree Star, Inc. USA).Cells were first gated by their morphology excluding dead cells byselecting DAPI viable cells.

AAV Generation and Use In Vitro and In Vivo:

Complete cDNA sequence of rat IL-34 containing Q81 (9), or GFP ascontrol, were positionned downstream a RSV promotor. Plasmids were firsttested in HEK293T cells transfected with lipofectamine reagent (LifeTechnologies, Carlsbad, Nouveau-Mexique). Cells were analysed for GFPand IL-34-myc expression 48 h later by FACS with anti-myc Ab. Then,plasmids were used to produce AAV vectors of serotype 8 (LTG platform,INSERM UMR 1089, Nantes). HEK293T cells were transduced with 10 000, 100000 to 1 000 000 MOI vector genome copies/cell of AAV-IL-34 or AAV-GFPand 5 000 MOI AdLacZ. 24 h later, cells were harvested and analysed forIL-34-Myc expression by FACS, and supernatent was tested for suppressionactivity on CD4+ Tcells responding to allogeneic pDCs, at a 1/10 and ⅕dilution. Recombinant AAV-IL-34 and AAV-GFP (4.5.10¹⁰, 1.10¹², and2.10¹² vector genomes/rat) vectors were injected i.v. in 4-weeks-oldrats one month before transplantation to allow optimal expression fromAAV vectors (23) Blood samples were taken for donor allospecificantibodies quantification.

Adoptive Cell Transfer:

Rat cells were sorted as previously described (5, 8) by FACS Aria (BDBiosciences, Mountain View, Calif.) by gating on TCRαβ-APC (R7/3),CD45RA-FITC (OX33), and CD11b/c-biotin-Streptavidine PECy7 (OX42)positive cells. Recipients that received splenocytes from IL34-treatedrats are defined as 1^(st) transferred and then iterative transfers weredefined as 2^(nd)- to 3^(rd) transferred. Total splenocytes (1.5×10⁸cells) and FACS Aria-sorted CD45RA⁺ B cells (6×10⁷), T cells (4×10⁷),CD11b/c⁺ cells (1.5×10⁷), CD4⁺CD25^(high) Tregs (4×10⁶) orCD8⁺CD45RC^(low) Tregs (4×10⁶) were adoptively transferred i. v. the daybefore heart transplantation into naive LEW-1A recipients that hadreceived 4.5 Gy of whole-body irradiation the same day.

Mixed Lymphocyte Reaction:

Naive Lewis 1A CD4⁺ T cells, naïve Lewis 1W pDC, and AdCD40Ig-treatedLewis 1A CD8⁺CD45RC^(low)Tregs subsets were sorted as previouslydescribed (16). Serum from AAV-IL-34-treated, AdCD40Ig-treatedrecipients and naïve rats were added in coculture to reach 3.12%, 6.25%,and 12.5% final concentration. Supernatent of transduced cells was addedto CD4⁺ T cells and pDC from 10% to 20% final concentration forsuppressive activity test. Rat IL-34, CD115 or M-CSF-blocking Ab orisotypic control were tested for blocking activity from 1.25 to 30 μg/mlin presence or not of CD8⁺CD40Ig Tregs. M-CSF protein (ab56288, ABCAM)was tested from 0.1 to 2 μg/ml. Proliferation of CFSE-labelled CD4⁺CD25⁻T cells was analyzed by flow cytometry 6 days later, by gating onTCR⁺CD4⁺ cells (R7/3-APC, Ox35-PECY7) among living cells (DAPInegative).

Sorted human CD4⁺CD25⁻ T cells were plated in triplicate with allogeneichuman T-depleted PBMCs in 200 μl of complete RPMI-1640 medium in roundor conic bottom 96-well plates, respectively, at 37° C. and 5% CO2.Human IL34 Ab was used at 50 μg/ml, and variable numbers of Tregs wereadded. Isotype control Ab were used at the highest concentrationdisplayed in the respective graph. M-CSF protein (ab56288, ABCAM) wastested from 0.1 to 2 μg/ml. Soluble human IL34 (eBiosciences) was addedat a concentration of 1, 2 or 5 μg/ml for the suppressive activity test.

Donor Specific Alloantibodies Quantification:

Donor spleens were digested by collagenase D, stopped with 400 μl EDTA0.1 mM, and red cells were lysed. Serum of recipients were added todonor splenocytes at a dilution ⅛, and incubated with either anti-ratIgG-FITC (Jackson ImmunoResearch Labs INC, Baltimore, USA), anti-ratIgG1 (MCA 194, Serotec), anti-rat IgG2a (MCA 278, Serotec), or anti-ratIgG2b (STAR114F, Serotec) and anti-Ms Ig-FITC(115-095-164, JacksonImmunoResearch). A FACS Canto (BD Biosciences, Mountain View, Calif.)was used to measure fluorescence, and data were analyzed using theFLOWJO software (Tree Star, Inc. USA). Geometric mean of fluorescence(MFI) of tested sera was divided by mean of 5 naive Lewis 1A sera MFI ascontrol.

Statistical Analysis:

One Way ANOVA Kruskal Wallis test and Dunn's posttest was used for PCRand coculture experiments, Two-Way ANOVA test and Bonferroni posttestswas applied for donor-directed antibodies, and splenocytes phenotypecharacterization, and Mantel Cox test was used to analyse survivalcurves.

Clodronate Liposomes In Vivo Treatment:

Clodronate liposomes for macrophage depletion were purchased from VrijeUniversity, The Netherlands (www.clodronateliposomes.org) and preparedas recommended (5041). Briefly, 2.5 ml of suspended solution wasadministered weekly intra-peritonealy from day −25 to day 3.

Quantitative RT-PCR:

Total RNA was isolated from cells using Trizol reagent (Invitrogen) oran RNeasy Mini Kit (Qiagen). RNA from macrophages was amplified withMessageAmpTMII aRNA Amplification Kit according to the manufacturerinstructions (Life Technologies) and reverse transcripted with randomprimers and M-MLV reverse transcriptase (Life Technologies). Real-timePCR was done using the Fast SYBR Green technology in a 20 μL finalreaction volume containing 10 μL of Master Mix 2× (Life Technologies),0.6 μL of primers (10 μM), 1 μL of cDNA and 8.4 of μl water. Thereaction was performed on the Applied Biosystems StepOne™ (LifeTechnologies). The thermal conditions were the following: 3 sec at 95°C., 30 sec at 60° C. and 15 sec at TM-5° C. with a final melting curvestage.

Results

IL-34 was Expressed by Splenic CD8⁺CD45RC^(low) Treg and the TolerantAllograft:

DNA microarray analysis of CD8⁺CD40Ig Tregs vs. naive CD8⁺CD45RC^(low)Tregs from spleen, highlighted IL-34 upregulation (among the mostupregulated genes) by CD8⁺CD40Ig Tregs, with a fold change of 4.05. Thisupregulation was confirmed by qPCR with >11 fold increase of IL-34 mRNAexpression in long-term splenic CD8⁺CD40Ig Tregs compared with naiveCD8⁺CD45RC^(low) Tregs (p<0.05, FIG. 1A).

Looking at whole organs, IL-34 mRNA was expressed endogenously in spleenand heart of naive animals (as observed by Lin et al. (18)) (FIG. 1B),and slightly decreased during acute allograft rejection in both spleenand graft (NT, FIGS. 1B and 1C). In correlation with our previousobservations that CD8⁺CD40Ig Tregs accumulated in the graft during thefirst week (16), IL-34 mRNA expression was significantly increased atday 5 in AdCD40Ig-treated graft and spleen (FIGS. 1B and 1C). Thissignificant increase was still detectable in AdCD40Ig-treated graft 120days after transplantation; however in the total spleen IL-34 mRNA levelhad return to normal.

To confirm the protein expression of IL-34, we labeled CD8⁺CD40Ig Tregswith a mouse anti-rat IL-34 antibody (Ab) that we generated. With thisAb, we confirmed the significant expression of IL-34 by CD8⁺CD40Ig Tregscompared to naive CD8⁺CD45RC^(low) Tregs (FIG. 1D).

Altogether, these results demonstrated for the first time that IL-34 canbe expressed by induced CD8⁺CD45RC^(low) Tregs, as well as toleratedallograft. Moreover, the early expression of IL-34 in graft and spleensuggest its early involvement in the inhibition of acute graft rejectionand thus the establishment of allograft tolerance. IL-34 expressed byCD8⁺CD45RC^(low) Treg, but not M-CSF, is involved in Treg-mediatedsuppression: We previously demonstrated that CD8⁺CD40Ig Tregs suppressanti-donor proliferation of CD4⁺ effector T cells in response toallogeneic pDCs ex vivo (16). In addition, we demonstrated theinvolvement of IFNγ and FGL2 in this process; however some suppressionremained after blockade of IFNγ and FGL2 inhibitory effect (15, 16). Toaddress whether IL-34 was involved in CD8⁺CD40Ig Treg suppression, wetested a neutralizing anti-IL-34 antibody in the suppressive MLR assay(FIG. 2A). The addition at increased concentration of blockinganti-IL-34 Ab resulted in a dose dependent increased of CD4⁺proliferation up to 59% reversal of CD8⁺ Tregs-mediated inhibition.

Given that IL-34 has similarities with M-CSF, that we observed asignificant expression of M-CSF by CD8⁺CD40Ig Tregs compared to naiveCD8⁺CD45RC^(low) Tregs (FIG. 6A), and compete for the same receptor (18,19), we wanted to address the possibility that M-CSF could play a rolein the inhibition of the proliferation of effector T cells. First ofall, we tested the suppressive potential of M-CSF in the MLR assaydescribed before. Interestingly, we observed that M-CSF efficientlysuppressed up in a dose dependent manner up to 93.5% of CD4⁺CD25⁻ Tcells proliferation (FIG. 6B), suggesting that M-CSF-mediatedsuppression, as for IL-34, acts through pDCs expressing the CSF1-R (20,21). However, the addition of a blocking anti-M-CSF Ab in co-culturesuppressive assays in the presence of CD8⁺ Tregs did not restore CD4⁺ Tcell proliferation, demonstrating that M-CSF was not involved inCD8⁺CD40Ig Treg-mediated suppression (FIG. 2B).

We next tested the involvement of CSF1-R, the only peripheral receptordescribed until now for IL-34 (18, 19) expressed bymonocytes/macrophages, cDCs and pDCs (20, 21). Thus, we used ananti-CSF1-R-blocking Ab that has been previously shown to inhibit M-CSFactions in both rats and mice (22). We demonstrated that blocking ofCSF1-R significantly abrogated CD8⁺ Treg-mediated suppression on CD4⁺ Tcell proliferation in presence of pDCs (FIG. 2C).

In conclusion, we demonstrated the involvement of IL-34/CFS1-Rinteractions, but not M-CSF/CSF1-R interactions, in the suppressiveeffect of CD8⁺CD40Ig Tregs.

Generation of an Adeno-Associated Viral (AAV) Vector for SustainedExpression of IL-34:

To further analyze the suppressive activity of IL-34, and sincerecombinant IL-34 rat cytokine was not commercially available anddifficult to produce for in vivo experiments, we generated a recombinantAAV vector encoding IL-34 rat molecule, as we have done for othermolecules in primates (23) and rats (24). In this vector, the rat IL-34cDNA was fused with a C-terminal Myc tag and both plasmid (pIIL-34) andlentivirus were first used to stably transfect or transduced HEK293 Tcell lines. IL-34 expression was indicated by flow cytometry for theMyc-tag. Myc staining was not detectable on untransfected or AAV-GFPtransduced HEK293 T cells, as well as on HEK293 T cells stained withisotypic control Ab. However, HEK293 T cells transfected with pIIL-34 ortransduced with AAV-IL-34 expressed strong amount of IL-34 protein in adose dependent manner, demonstrating the secretion of IL-34 and thefunctionality of the vector.

We then tested the suppressive potential of AAV-IL34 transduced HEK293 Tcells culture supernatant (FIG. 3A) and sera of AAV-IL34 treated-rats(FIG. 3B), that both contain high amount of IL-34 protein, as shownpreviously. We observed that both supernatant from AAVIL-34-transducedcells and sera from AAV-IL34 treated rats significantly inhibited theproliferative response of CD4⁺ effector T cells stimulated by allogeneicpDCs (in a dose dependent manner) in comparison to controls (FIGS. 3Aand 3B).

Altogether, these results demonstrated the functionality of the vectorand the suppressive efficacy of IL-34 in inhibiting effector T cellsproliferation, thus suggesting its potential in vivo in transplantation.

Therapeutic Effect of IL-34 in Allograft Tolerance Induction:

To further determine the suppressive potential of IL-34 in vivo as atherapeutic strategy, we treated recipients with either AAV-IL-34 1.10¹²vg/rat or a control non-coding AAV, i.v. one month beforetransplantation. Such treatment with IL-34 alone resulted in asignificant prolongation of allograft survival (mean survival time32.6±7.8 days) vs. controls injected with non-coding AAV (14.2±1.8 days)or untreated recipients (7.8±0.6 days) (FIG. 4). To improve allograftsurvival, recipients were then treated with a suboptimal dose ofrapamycin (during 14 days) in addition to the AAV vector. 14 days ofrapamycin alone did not significantly extend allograft survival (FIG. 4,black circle). In contrast, we observed an indefinite allograft survivalin 75% of the recipients that had received the combined therapy AAV-IL34and rapamycine compared to controls (p<0.001, FIG. 4). Analysis of graftof long-surviving recipients for signs of chronic rejection revealed acomplete absence of vascular lesions i.e. normal vessel structure andabsence of leukocyte infiltration in the myocardium in all recipientsanalyzed. In addition, analysis of presence of anti-donor antibodies inthe sera of long-surviving recipients revealed a significant inhibitionof total IgG, IgG1, IgG2a and IgG2b anti-donor Abs versus recipienttreated with the non coding AAV.

Altogether, we were able to demonstrate for the first time that IL-34 isa valuable therapeutic strategy for tolerance induction in combinationwith rapamycin and resulted in abrogation of all allogeneic immuneresponses.

IL-34 Potently Induces Regulatory T Cells Capable of InfectiousTolerance:

As demonstrated above, IL34 is produced specifically by CD8⁺CD40IgTregs. We next assessed whether regulatory cells were induced in thecontext of IL34-treatment and involved in the long-term allograftsurvival generated by AAV-IL34 and rapamycin combination. To do so, weperformed adoptive cell transfer experiments using splenocytes oflong-surviving recipients into naive grafted irradiated recipients, aswe have done before (15). First adoptive transfer of 1,5.10⁸ splenocytesinto secondary naive grafted irradiated recipients resulted insignificant prolongation of allograft survival of 60% of the recipients(FIG. 5A), demonstrating that IL-34 efficiently induces regulatorycells. We investigated the anatomopathological status of the graft offirst adoptively transferred long-term splenocytes recipients andobserved a complete absence of vascular lesions and obstructions (i.e.no signs of chronic rejection). We then determined whether thisprolongation of allograft survival can be serially transferred to secondand third recipients and we observed that tolerance can be seriallytransferred at least 3 times into naive grafted irradiated recipients(FIG. 5, 2^(nd) and 3^(rd) Transfer).

Given that IL-34 was recently described to induce regulatory macrophages(25), we investigated the regulatory population allowing serial adoptivetolerance transfer including macrophages. To do so, we purifiedsub-populations of the different main subsets (B cells, T cells andmacrophages) from tolerant recipients treated with IL34 and performedadoptive cell transfer into naive irradiated grafted recipients (FIG.5B). To our surprise, we observed that tolerance transfer was achievedonly with T-cell transfer, and not macrophages as suggested by others,demonstrating for the first time that IL-34 can induce regulatory Tcells and that in our model, macrophages were not potent enough toinhibit acute allograft rejection. However, regulatory T cells do notexpress the IL-34's receptor suggesting that macrophages are a necessaryintermediate in regulatory T cells functions. To further determine whichTreg population (i.e. CD4⁺CD25^(high) or CD8⁺CD45RC^(low) T cells) couldgive tolerance to newly grafted recipients, we sorted CD4⁺CD25^(high)and CD8⁺CD45RC^(low) Tregs and performed adoptive cell transfer (FIG.5B). We observed that both adoptive transfers of CD4⁺CD25^(high) andCD8⁺CD45RC^(low) Tregs resulted in 50% of long-term allograft survivalin recipients, suggesting that both populations of Tregs had beenequally potentiated by the IL34-modified macrophages.

Altogether, these in vivo results demonstrate that efficient Tregs aregenerated following IL34-treatment in the context of reducedinflammation and transplantation, and that those Tregs can induce serialtolerance in a dominant fashion.

Treg Induction is Mediated by IL34 Modified-Macrophages Infiltrating theGraft:

In an attempt to further identify the role of IL-34-induced macrophagesin the induction of tolerance, we characterized the effect of IL34 onmacrophages in the context of tolerance induction to an allograft. Wefirst sorted macrophages from spleen, blood and graft of AAV-IL34treated recipients at day 15 following transplantation (i.e. day 45post-AAV injection) and macrophages from naive rats and analyzed by qPCRa number of genes (FIG. 7). Interestingly, we observed that macrophagesin the graft from AAV-IL34-treated recipients strongly upregulatedarginase 1 and inducible NO synthase (iNOS), both implicated in themetabolism of essential amino acids and described as common mechanism ofimmunoregulation of suppressive macrophages by limiting proliferation ofT lymphocytes (34), compared to naive macrophages. We also observed anincreased expression of CD14 and a decreased expression of CD23, CD86and IL10, mostly in the graft, but also in the spleen and the blood ofAAV-IL34 treated macrophages compared to naive macrophages. Finally, weobserved no significant differences for CD16, CD32, ILL TGFβ and TNFαexpression. It is also interesting to note that there were significantdifferences between macrophages located in the blood versus the graft,suggesting that IL34-modified regulatory macrophages migrate and locatein the graft quickly after transplantation. We further depleted themacrophages populations using clodronate-loaded liposomes from day −25before transplantation to day 3 after transplantation and treatedsimultaneously with IL34 or non-coding AAV plus sub-optimal dose ofrapamycin during 10 days, as previously described by others.Unfortunately, this therapy resulted in itself in allograft survival andcould not be used to reveal the role of IL-34-induced macrophages. Bydoing so, AAV-IL34 injected 30 days before transplantation could not actthrough macrophages that were depleted at that same time. Significantly,by the time the liposome depletion started, most of the AAV serotype 8had been integrated in the hepatocytes from the liver. We observed thatrecipients treated with clodronate-loaded liposomes rejected their graftmore quickly after transplantation compared to the control group,demonstrating that macrophages are essential in tolerance induction byIL34 (FIG. 8).

IL34 Possesses a Strong Suppressive Potential:

As we suspected a suppressive potential of IL34 in human, we addeddifferent doses of soluble human IL34 to a MLR whereCD4⁺CD25⁻CFSE-labeled effector T cells were cultured in presence ofT-cell depleted allogeneic PBMCs as APCs (FIG. 9A). We observed asignificant dose-dependent inhibition of effector T-cell proliferationin the presence of IL34, thus confirming the suppressive potential ofIL34 on anti-donor immune response. Finally, to demonstrate theinvolvement of IL34 in CD4⁺CD25^(high)CD127^(low) and CD8⁺CD45RC^(low)Treg-mediated suppressive activity on anti-donor immune responses, weadded either anti-human IL34 blocking Ab or a control isotype Ab to aMLR where CFSE-labeled CD4⁺CD25⁻ effector T-cell proliferation in thepresence of allogeneic T-depleted PBMCs is inhibited by Tregs (FIG. 9B).We observed that blocking IL34 significantly reverted Treg-mediatedsuppression for both CD4⁺ and CD8⁺ Tregs compared with isotype controlAb, demonstrating the key role of IL34 in Tregs' suppressive activity.

Altogether, these data prove the relevance of our findings and providethe proof of concept of IL34 as a Treg-specific protein and a potentialtherapeutic target in manipulating the anti-donor immune response.

Discussion

The biological relevance of IL-34 remains to date largely unknown andcontroversial. The current understanding of the role of IL-34 was mostlydriven by study on pathological situations were IL-34 was found to exertinflammatory functions, such as M-CSF. Various studies have shown thatM-CSF administration increases inflammation in a model ofcollagen-induced RA (26) and that IL-34 correlates with severity ofsynovitis, inflammation in a model of RA (13) and can be induced byTNFα, as M-CSF (27). Furthermore, both IL-34 and M-CSF induceproinflammatory cytokines as IL-6, IP10/CXCL10, IL-8/CXCL8, MCP1/CCL2(28). In contrast with these studies, it has also been shown that M-CSFand more recently IL-34, alone or in combination with other cytokines,can induce regulatory macrophages (25, 29-32). In transplantation, ithas been demonstrated that M-CSF pre-treatment of mice expandmacrophages and inhibit GVHD (33). In addition, combination of M-CSF andIFNγ differentiate monocytes in regulatory macrophages capable toprolong heart allograft survival in an iNOS dependent manner (34). Thesestudies underlie the paradoxical role of IL-34. In our study, in anattempt to unravel the complex mechanisms of tolerance induction intransplantation, we provide evidences, for the first time, of theunexpected properties of IL-34 as a master regulator of immune responsesand tolerance. We also provide the first proof that IL-34 can beexpressed by tolerated allografts and CD8⁺CD45RC^(low) Tregs, and mostimportantly can induce potent regulatory T cells.

We previously demonstrated that treatment of cardiac graft recipientswith an adenovirus encoding CD40Ig lead to indefinite allograft survivalin 93% of the recipients, and that this acceptance was mediated byCD8⁺CD45RC^(low) Treg in a IFNγ, IDO and FGL2 dependent manner. We morerecently demonstrated that CD8⁺CD45RC^(low) Treg recombined a biasedrestricted Vβ11 repertoire to recognize a dominant MCH class II derivedpeptide, and that this peptide induces regulatory Tregs and inducestolerance (17). In the present specification, we show that IL-34 wasexpressed at high level by tolerated grafts of AdCD40Ig-treatedrecipients, and importantly, also by splenic CD8⁺CD45RC^(low) Tregs fromthe same recipients. Furthermore, CD8⁺CD45RC^(low) Tregs mediatedsuppression can be partially abrogated by blockade of IL-34. Thus, IL-34possesses immunosuppressive properties that have never been studieduntil now, and acts in synergy with FGL2, IDO and IFNγ in the CD40Igmodel of suppression mediated by CD8⁺CD45RC^(low) Tregs (16).

We also demonstrated that this property was specific of IL-34 since weobserved that M-CSF was not involved in this model. Accumulatingevidences suggest that IL-34 and M-CSF exhibit specific andnon-redundant properties. This is underlined by structural analysiscomparison showing that IL-34 and M-CSF bind differently to CD115 (11,19). The identification more recently of a second distinct receptor forIL-34 reinforces this interpretation (10). Very recently,IL-34-deficient mice have been generated and showed disappearance ofcertain cell subsets such as Langerhan's cells and microglia (12),effects that had not been observed in M-CSF KO mice and demonstrating,not only a different temporal and spatial expression role, but alsodifferent functional effects for IL-34 vs. M-CSF.

The therapeutic value of this molecule was evidenced with the generationof an AAV encoding IL-34. With this vector, we were able to show for thefirst time the potent immunosuppressive properties of IL-34 in vitroand, most importantly in vivo where we obtained indefinite allograftsurvival in 80% of the recipients when combined with a sub-optimal doseof rapamycin. We also demonstrated that such therapy resulted inabrogation of all allogeneic immune responses and the induction oftolerance. Previous studies demonstrated that both M-CSF and IL-34 candifferentiate monocytes in regulatory macrophages (25, 34) and theregulatory macrophages induced in vitro by M-CSF and IFNγ can be used invivo to prolong heart allograft survival in mice (34). Another studydemonstrated in mice that administration of M-CSF before transplant canexpand macrophages and thus limit donor T cell expansion and GVHD (33).Chen et al. showed in mice treated with soluble IL-34 protein anincrease of the CD11b⁺ population (35). Moreover, other studies noticeda decrease in pDC and cDC in CSF-1-deficient osteopetrotic mice (21),and a CFS1-induced increase in DC number (20). Surprisingly, and incontrast with other study, in vivo IL-34 tolerogenic effect followingadministration was mediated by regulatory T cells. Indeed, wedemonstrated that the tolerance obtained in AAV-IL-34-treated recipientscould be transferred in newly grafted irradiated recipients for at least3 generations and that this effect was mediated by Tregs, but, despitethe increase observed for the CD11b⁺ cell population, not bymacrophages. However, since Tregs do not express IL-34's receptor, wecan hypothesize that IL-34 mediated its effect on Tregs throughmacrophages as it has been shown in the literature that regulatorymacrophages can anergizing CD4 effector T cells (36), converting T cellsin Tregs (37) or inhibiting other APCs presentation (38). We could notconclude that IL-34 induces regulatory macrophages in our model, butthese are necessary intermediate in IL-34 induced Tregs. These resultshighlight the functional differences of IL-34 and M-CSF and thecontroversy on this topic as several studies showed the pro-inflammatoryrole of MCSF that increases macrophage proliferation and accumulation inrejected renal allograft (39).

In conclusion, we described here the role in transplantation toleranceof a new cytokine, IL-34, and we revealed its potential as a therapy intransplantation or as a biomarker associated with better prognosis intransplantation, but also by extension in other diseases. We alsodemonstrated for the first time that this cytokine can be produced byCD8⁺ Tregs and can in turn, induce Tregs capable of tolerance inductionin a dominant manner, opening new possibilities in the generation ofTregs transferrable to the human setting.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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1. A method of i) inducing immune tolerance, ii) preventing or reducingtransplant rejection, or iii) preventing or treating autoimmunediseases, unwanted immune responses against therapeutic proteins andallergies in a patient in need thereof, comprising administering to thepatent a therapeutically effective amount of an Interleukin-34 (IL-34)polypeptide or a polynucleotide encoding the Interleukin-34 (IL-34)polypeptide.
 2. (canceled)
 3. The method according to claim 1, whereinthe transplant rejection is cardiac allotransplant rejection. 4.(canceled)
 5. The method according to claim 1, wherein the IL34polypeptide has a sequence comprising or consisting of SEQ ID NO: 1 or asequence having at least 80% identity to SEQ ID NO:
 1. 6. Apharmaceutical composition or a kit-of-part composition comprising anisolated IL-34 polypeptide or a polynucleotide encoding therefor and animmunosuppressive drug.
 7. The pharmaceutical composition or akit-of-part composition according to claim 6, wherein theimmunosuppressive drug is selected from the group consisting ofcytostatics; alkylating agents; antimetabolites; therapeutic antibodies;calcineurin inhibitors; glucocorticoids and mycophenolate mofetil. 8.The pharmaceutical composition or kit-of-part composition according toclaim 7, wherein the immunosuppressive drug is rapamycin (sirolimus). 9.A method of preventing or treating of transplant rejection, autoimmunediseases, unwanted immune response against therapeutic proteins andallergies in a patient in need thereof, comprising administering to thepatient a therapeutically effective amount of pharmaceutical compositionaccording to claim
 6. 10. An in vitro method for determining whether apatient is at risk of transplant rejection, autoimmune diseases, or anunwanted immune response against therapeutic proteins or allergies,comprising a step of determining an expression level of IL-34 in abiological sample obtained from said patient, wherein the presence ofIL-34 is indicative of a reduced risk of transplant rejection,autoimmune diseases, unwanted immune response against therapeuticproteins or allergies.
 11. The method according to claim 10, wherein theexpression level of IL-34 is determined by measuring the concentrationof IL-34 in said biological sample.
 12. The method according to claim10, wherein the biological sample is a blood sample (serum or plasmasample).
 13. A method for adjusting the immunosuppressive treatmentadministered to a patient in need thereof, comprising (i) determiningthe risk of transplant rejection, autoimmune diseases, or an unwantedimmune response against therapeutic proteins or allergies according tothe method of claim 10, and (ii) based on results obtained in saiddetermining step, adjusting the immunosuppressive treatment.
 14. Thepharmaceutical composition or a kit-of-part composition according toclaim 7, wherein the cytostatic is mammalian target of rapamycin (mTOR)inhibitor or rapamycin.
 15. The pharmaceutical composition or akit-of-part composition according to claim 7, wherein the alkylatingagent is cyclophosphamide.
 16. The pharmaceutical composition or akit-of-part composition according to claim 7, wherein the antimetaboliteis azathioprine, mercaptopurine or methotrexate.
 17. The pharmaceuticalcomposition or a kit-of-part composition according to claim 7, whereinthe therapeutic antibodies are anti-CD40L monoclonal antibodies,anti-IL-2R monoclonal antibodies, anti-CD3 monoclonal antibodies,anti-lymphocyte globulin (ALG) or anti-thymocyte globulin (ATG).
 18. Thepharmaceutical composition or a kit-of-part composition according toclaim 7, wherein the calcineurin inhibitor is cyclosporine.
 19. Themethod of claim 12, wherein the blood sample is serum or plasma.